CN115975962A - Net path product directed synthesis - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to the directional synthesis of network pathway products. Compared with the conversion of steroidal substrates by using natural microbial endogenous pathways, the present invention utilizes the reconstruction of artificial heterologous network pathways under the yeast chassis and rationally combines pathway component proteins to achieve directional synthesis of node products. Further improvements in pathway conversion efficiency can be achieved by combining homologous proteins of pathway components expressing different catalytic specificities. At the same time, the artificial pregnenolone synthesis pathway is constructed under the microbial chassis, and by introducing specific downstream androstenedione synthesis pathway components, the directional synthesis of androstenedione pathway node compounds is achieved using simple carbon sources as substrates.

Description

Directed synthesis of network pathway products

Technical Field

The invention relates to the field of biotechnology, in particular to the directional synthesis of network path products.

Background Art

In chordates, the steroid hormone synthesis pathway is composed of numerous non-specific catalytic enzymes, which can modify a variety of structurally similar substrates. Therefore, enzymes often share multiple substrates. The cross-reactions between pathways ultimately make the steroid synthesis pathway present a complex network catalysis, which greatly increases the difficulty of microorganisms to heterologously synthesize intermediate node compounds in the network pathway.

Steroid hormones are a class of tetracyclic aliphatic hydrocarbons with a cyclopentane polyhydrogen phenanthrene nucleus as the basic structure. There are many types of such compounds. At present, there are more than 400 steroid drugs produced in the world. They are widely used for their anti-inflammatory, anti-allergic and endocrine regulating effects. By 2017, the global sales of steroid hormone drugs reached 100 billion US dollars, making it the second largest class of chemical drugs. The "pregnenolone-androstenedione" synthesis pathway composed of CYP17-3β-HSD is located at the intersection of the vertebrate steroid synthesis pathway, which contains the key precursors of many steroid hormone drugs (pregnenolone, progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, DHEA, androstenedione, testosterone).

The development of the production process of steroid hormone intermediates has gone through stages such as plant extraction saponin method, chemical total synthesis method, semi-synthesis method, and new microbial synthesis method. Plant extraction method and microbial transformation method are the main methods for producing steroid hormone drugs. However, the animal and plant extraction method has high costs, limited sources, and is accompanied by environmental pollution. Chemical total synthesis of steroid molecules involves limitations such as long processes, complex reactions, and environmental pollution.

Compared with steroids isolated from animal sources, microbial synthesis of steroids has a low risk of viral/prion contamination. Microbial synthesis of steroids can also avoid the cumbersome reaction steps involved in chemical synthesis. In previous studies, Saccharomyces cerevisiae, Escherichia coli, and Yarrowia lipolytica have all been used in the study of pregnenolone or other steroid synthesis. In the experiment of Saccharomyces cerevisiae, de novo synthesis of pregnenolone and downstream product hydrocortisone were achieved from glucose as substrate, and Saccharomyces cerevisiae, Escherichia coli, and Yarrowia lipolytica achieved the synthesis of pregnenolone from biotransformation of sterol substrates. The biotransformation of pregnenolone can be achieved by introducing the P450scc catalytic system into Saccharomyces cerevisiae or Escherichia coli and adding sterols, the direct substrate of the P450scc catalytic system, to the culture environment. Sterol feeding in the culture environment of Saccharomyces lipolytica diploid chassis cells heterologously expressing the P450scc system and the P450c17 system can enable them to use sterols as substrates for biotransformation and synthesis of pregnenolone or 17α-hydroxypregnenolone. However, the strong hydrophobicity of steroids, the poor robustness of commonly used microorganisms Mycobacterium, and the high cost of sterilization result in low microbial conversion efficiency.

Synthetic biology provides a new method of microbial synthesis. It can artificially construct functional microorganisms with heterologous synthesis pathways, which can produce steroid hormone drugs of specific structures with low energy consumption, high efficiency and environmental friendliness. It can produce specific steroid compounds with only glucose, glycerol and other single carbon sources. In 1998, Catherine Duport and others achieved the synthesis of campesterol by knocking out the inherent gene erg5 of Saccharomyces cerevisiae and introducing the exogenous gene DHCR7, providing the synthetic precursor of pregnenolone; and then introduced the bovine P450scc catalytic system and 3β-HSD to achieve the de novo synthesis of progesterone with glucose as the substrate. In 2019, based on the high-yield campesterol chassis, our research group constructed a lipolytic yeast engineered bacterium that synthesizes pregnenolone from scratch through enzyme source screening and promoter assistance. In 2003, Florence Ménard Szczebara et al. knocked out ATF2 based on the high-progesterone-producing Saccharomyces cerevisiae chassis, and introduced CYP17A1, CYP21A1, and CYP11B1, successfully achieving the de novo synthesis of hydrocortisone. In 2019, patent US10400261B2 integrated multiple copies of hydrocortisone pathway genes based on Saccharomyces cerevisiae to obtain a high-yield strain of hydrocortisone.

Corticosteroids, androgens and estrogens have been widely used in the medical field. The synthesis of these downstream products in the animal steroid hormone synthesis pathway requires the conversion of Δ ⁵ - to Δ ⁴ -type steroids (Δ ⁵ - and Δ ⁴ -: olefinic double bonds at positions 5.6 and 4.5, respectively), which uses pregnenolone (P5) as a substrate and is catalyzed by 3β-hydroxysteroid dehydrogenase (3β-hsd) and 17α-hydroxylase/17,20-lyase (Cyp17). Among them, CYP17 uses Δ ⁵ - and Δ ⁴ -steroids as substrates to carry out 17α-hydroxylation modification. At the same time, under the condition of the participation of cytochrome b5 (Cyb5), CYP17 will show stronger 17,20-lyase activity (C17-C20 bond cleavage), and CYP17 can; while 3β-HSD has isomerase activity, which can convert Δ ⁵ -steroids into corresponding Δ ⁴ -isomers and realize the migration of metabolic flux from Δ ⁵ -pathway to Δ ⁴ -pathway. However, in the process of synthesizing Δ ⁴ -steroids using pregnenolone as substrate, since CYP17 and 3β-HSD share multiple substrates, 17α-hydroxylation, 17,20-cleavage and Δ ⁵ -Δ ⁴ isomerization do not occur in strict sequence, which brings great challenges to the heterologous directed synthesis of target Δ ⁴ -steroids.

Steroid hormones are often toxic to microorganisms. In an environment containing steroid compounds, endogenous microbial metabolism can modify (hydroxylate, etc.) the exogenous steroid nucleus to reduce its cytotoxicity. Taking advantage of this property, people use endogenous metabolism of microorganisms such as Mycobacterium as a medium to achieve the synthesis of target steroids through biotransformation using sterols and steroid intermediates as substrates.

The traditional use of microbial endogenous metabolism to biotransform steroid substrates to synthesize target steroids has the following disadvantages: ① The strong modification activity of the traditional biotransformation microbial chassis on the steroid parent nucleus may not be suitable for the synthesis of specific target steroid compounds, and may compete with the target synthesis pathway for steroid substrates, resulting in by-product accumulation and substrate loss. Therefore, it is more advantageous to use the Yarrowia lipolytica chassis with a relatively pure and clear metabolic background. ② In the endogenous modification pathways of steroids in traditional biotransformation microorganisms such as Mycobacterium, many key catalytic reaction-related genes are still unclear, which greatly increases the difficulty of path modification to achieve the directed synthesis of intermediate node products during the biotransformation process.

Summary of the invention

In view of this, the present invention provides a network-pathway product-directed synthesis.

The present invention provides directional synthesis of network pathway products. Compared with the conversion of steroid substrates by natural microbial endogenous pathways, the present invention uses the reconstructed artificial heterologous network pathway under the yeast chassis and rationally combines the pathway component proteins to achieve directional synthesis of node products. By combining and expressing homologous proteins of pathway components with different catalytic specificities, the pathway conversion efficiency can be further improved. At the same time, by constructing an artificial pregnenolone synthesis pathway under the microbial chassis and introducing specific downstream androstenedione synthesis pathway components, the directional synthesis of androstenedione pathway node compounds using simple carbon sources as substrates can be achieved.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The present invention provides the use of any of the following expressions in the synthesis of steroidal compounds and/or steroidal hormone drugs:

(I), CYP17A1 and POR from Equus caballus, Ovis aries, Mesocricetus auratus or Xenopus laevis; and/or

(II), CYB5 from Equus caballus, Ovis aries, Mesocricetus auratus or Sus scrofa; and/or

(III), 3β-HSD from Mus musculus, Bos taurus, Vaccinia virus, Arabidopsisthaliana, Mycobacterium tuberculosis, Homo sapiens (type I), Homo sapiens (type II), Homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation); and/or

(IV), mCYP11A1 from Sus scrofa.

In some specific embodiments of the present invention, the CYP17A1, POR, CYB5, 3β-HSD and/or mCYP11A1 are synthesized by adding sequence 1 and/or sequence 2 through codon optimization;

The codon optimization was performed using Yarrowia lipolytica;

The sequence 1 adds a 5' end, and the sequence 1 comprises:

(I), the nucleotide sequence shown in SEQ ID NO: 1; or

(II) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I) but differing from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

(III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (I) or (II); or

(IV) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (I) to (III);

The sequence 2 adds a 3' end, and the sequence 2 includes:

(I), the nucleotide sequence shown in SEQ ID NO: 2; or

(II) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I) but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

(III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (I) or (II); or

(IV) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (I) to (III);

The steroids include: pregnenolone, progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, dehydroepiandrosterone, androstenedione and/or testosterone;

In the conversion experiment with pregnenolone (P5) as the substrate, the one showing the strongest pregnenolone conversion efficiency was Vv_3β-HSD; the conversion efficiency was 6.8%; the ones showing the second strongest catalytic efficiency included type I human and mutant 3β-HSD; the catalytic efficiency was 4.6-4.8%;

In the conversion experiment with 17-hydroxypregnenolone (17OHP5) as the substrate, the ones that showed strong conversion efficiency included type I human 3β-HSD and mutant 3β-HSD, type II human 3β-HSD and mutant 3β-HSD; the type I human 3β-HSD had the highest progesterone conversion rate of 3.1%;

Taking dehydroepiandrosterone (DHEA) as a substrate, the ones showing stronger catalytic activity include type II human, bovine and mycobacterium-derived 3β-HSD; the conversion efficiency of type II human 3β-HSD is 10.5%.

The present invention also provides a module, including any of the following expressions:

(I), CYP17A1 and POR from Equus caballus, Ovis aries, Mesocricetus auratus or Xenopus laevis; and/or

(II), CYB5 from Equus caballus, Ovis aries, Mesocricetus auratus or Sus scrofa; and/or

(III), 3β-HSD from Mus musculus, Bos taurus, Vaccinia virus, Arabidopsisthaliana, Mycobacterium tuberculosis, Homo sapiens (type I), Homo sapiens (type II), Homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation); and/or

(IV), mCYP11A1 from Sus scrofa.

Based on the above research, the present invention also provides a plasmid, comprising the expression element.

The present invention also provides a host, comprising the plasmid.

In some specific embodiments of the present invention, the host comprises one or more of module one, module two, module three, module four, module five, module six, module seven, module A, module B, module C or module D:

The module one comprises CYP17A1 and POR derived from Equus caballus, Ovis aries, Mesocricetus auratus or Xenopus laevis; the module one comprises an IntD integration site and/or a Leu2 tag with LoxP sites at both ends; and/or

The module 2 comprises CYB5 derived from Equus caballus, Ovis aries, Mesocricetus auratus or Susscrofa; the module 2 comprises an IntB integration site and/or a Ura3 tag; and/or

The module three comprises 3β-HSD derived from Mus musculus, Bos taurus, Vaccinia virus, Arabidopsis thaliana, Mycobacterium tuberculosis, Homo sapiens (type I), Homo sapiens (type II), Homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation); the vector connected to the module three comprises pINA1269-Nat; and/or

The module four includes CYP17A1 and POR derived from Equus caballus; the module four includes an IntF integration site and/or a Leu2 tag with LoxP sites at both ends; and/or

The module five comprises: CYP17A1 and POR derived from Ovis aries; the module five knocks out the Leu2 tag; and/or

The module six comprises: 3β-HSD derived from Homo sapiens (type II); the module six comprises an IntC integration site and/or a Leu2 tag with LoxP sites at both ends; and/or

The module seven comprises: CYP17A1 and POR derived from Mesocricetus auratus, 3β-HSD derived from Homosapiens (type II) and 3β-HSD derived from Vaccinia virus; the CYP17A1 and POR comprise an IntD integration site and/or a Leu2 tag with LoxP sites at both ends; the vector connected to the 3β-HSD derived from Vaccinia virus comprises pINA1269-Nat; the vector connected to the 3β-HSD derived from Homo sapiens (type II) comprises pUC57-Kan-Simple; and/or

The module A comprises: CYP17A1 and POR derived from Equus caballus, Ovis aries, Mesocricetus auratus or Xenopus laevis; the module A comprises an IntD integration site and/or a Leu2 tag with LoxP sites at both ends; and/or

The module B comprises: CYB5 derived from Equus caballus, Ovis aries or Mesocricetus auratus; the module B comprises an IntB integration site and/or a Ura3 tag; and/or

The module C comprises: 3β-HSD derived from Mus musculus, Bos taurus, Vaccinia virus, Arabidopsis thaliana, Mycobacterium tuberculosis or Homo sapiens (type II) and mCYP11A1 derived from Sus scrofa; the vector connected to the module C comprises pINA1269; and/or

The module D comprises: CYP17A1 and POR derived from Ovis aries and Mesocricetus auratus; the module D comprises an IntF integration site and/or a Leu2 tag with LoxP sites at both ends.

In some specific embodiments of the present invention, the construction of module one includes: splicing the left arm of the IntD integration site and terminator 1; splicing the terminal sequence of terminator 2, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, and the right arm of the IntD integration site to obtain IntD-L and IntD-R; connecting CYP17A1 and POR with the expression module after BsmBI digestion to obtain an integration plasmid, assembling the CYP17A1 and POR modules with the same species source with pUC18H to obtain an integration plasmid, and obtaining module one after enzyme digestion;

The terminator 1 is the Saccharomyces cerevisiae GPM1t terminator; the terminator 2 is the Saccharomyces cerevisiae FBA1t;

The splicing method includes OE-PCR; the expression modules include TEF1inp-LIP2t-GPDt, GPDt-TEF1inp-OCT1t-FBA1t; the sources of the CYP17A1 and POR modules include Equus caballus, Ovis aries, Mesocricetus auratus or Xenopus laevis; the pUC18H is pUC18H digested with IntD-L, IntD, and HincII; the assembly method includes Gibson; and the enzyme digestion includes NotI digestion.

In some specific embodiments of the present invention, the construction of module 2 includes: splicing the left arm of the IntB integration site, the tag, the right arm of the IntB integration site, the promoter, the terminator and CYB5 to obtain an integration plasmid, and obtaining module 2 after enzyme digestion;

The tag includes the auxotrophic uracil tag Ura3; the promoter includes TEF1in; the terminator includes ACOt; the CYB5 source includes Equus caballus, Ovis aries, Mesocricetus auratus or Susscrofa; the splicing method includes Gibson; and the enzyme digestion includes NotI digestion.

In some specific embodiments of the present invention, the construction of module three includes: 3β-HSD is integrated into a vector by assembly, sequence 3 is introduced at the 5' end of the above gene by PCR reaction, and sequence 4 is introduced at the 3' end of the gene, and after linearization of the vector assembly, a recombinant plasmid is integrated, i.e., module three;

The 3β-HSD source includes Mus musculus, Bos taurus, Vaccinia virus, Arabidopsisthaliana, Mycobacterium tuberculosis, Homo sapiens (type I), Homo sapiens (type II), Homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation);

The vector includes pINA1269-Nat;

The sequence 3 comprises:

(I), the nucleotide sequence shown in SEQ ID NO: 3; or

(II) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I) but different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

(III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

(IV) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (I) to (III);

The sequence 4 comprises:

(I), the nucleotide sequence shown in SEQ ID NO: 3; or

(II) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I) but differing from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

(III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (I) or (II); or

(IV) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (I) to (III);

The enzymes used for linearization include BamHI and KpnI;

The 3β-HSD derived from Homo sapiens (type I) has a higher activity and uses DHEA as a major substrate, while the 3β-HSD derived from Homo sapiens (type II) tends to use P5 and 17OHP5 as substrates.

In some specific embodiments of the present invention, the construction of module four includes: splicing the left arm of the IntF integration site, pUC18H, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, the right arm of the IntF integration site, CYP17A1 and POR (the method is the same as that of module one) to obtain a fragment containing restriction sites at both ends, and obtaining module four after restriction digestion;

The pUC18H is pUC18H digested with HincII;

The CYP17A1 and POR sources include Equus caballus;

The splicing method includes Gibson; the restriction site includes NotI; and the restriction enzyme cutting includes NotI enzyme cutting.

In some specific embodiments of the present invention, the construction of module five includes: knocking out the Leu2 screening marker of SyBE_Yl2091004 to obtain SyBE_Yl2091004 without the Leu2 tag;

The knockout method includes the Cre-loxP system.

In some specific embodiments of the present invention, the construction of module six includes: splicing the left arm of the IntC integration site, the artificial promoter hp8d, Hs_3β-HSD2, the Yarrowia lipolytica terminator OCTt, the leucine nutritional screening label Leu2 with LoxP sites at both ends, and the right arm of the IntC integration site by the OE-PCR method to obtain a fragment containing NotI restriction sites at both ends; the above fragment is connected with the plasmid pUC57-Kan-Simple linearized with HindIII to obtain an integration plasmid.

In some specific embodiments of the present invention, the construction of module seven includes: simultaneously integrating module one expressing CYP17A1-POR from golden hamster (Mesocricetus auratus), module six, and module three expressing Vv_3β-HSD into ATCC201249.

In some specific embodiments of the present invention, the construction of module A includes: the construction of module A includes: the left arm of the IntD integration site, the terminator 1 is spliced; the terminal sequence of terminator 2, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, and the right arm of the IntD integration site are spliced to obtain IntD-L and IntD-R; CYP17A1 and POR are connected to the expression module after BsmBI enzyme digestion, and the CYP17A1 and POR modules with the same species source are assembled with IntD-L, IntD-R, and pUC18H to obtain an integration plasmid, and module A is obtained after enzyme digestion;

The terminator 1 is the Saccharomyces cerevisiae GPM1t terminator; the terminator 2 is the Saccharomyces cerevisiae FBA1t;

The splicing method includes OE-PCR; the expression modules include TEF1inp-LIP2t-GPDt, GPDt-TEF1inp-OCT1t-FBA1t; the sources of the CYP17A1 and POR modules include Equus caballus, Ovis aries, Mesocricetus auratus or Xenopus laevis; the pUC18H is pUC18H digested with IntD-L, IntD, and HincII; the assembly method includes Gibson; the digestion includes digestion with NotI;

The integration plasmids include pIntD-Oa_CYP17-POR, pIntD-Ma_CYP17-POR, pIntD-Ec_CYP17-POR, and pIntD-Xl_CYP17-POR.

In some specific embodiments of the present invention, the construction of module B includes: splicing the left arm of the IntB integration site, the tag, the right arm of the IntB integration site, the promoter, the terminator and CYB5 to obtain an integration plasmid, and obtaining module B after enzyme digestion;

The tag includes the auxotrophic uracil tag Ura3; the promoter includes TEF1in; the terminator includes ACOt; the CYB5 source includes Equus caballus, Ovis aries or Mesocricetus auratus; the splicing method includes Gibson; and the enzyme digestion includes NotI digestion.

In some specific embodiments of the present invention, the construction of module C includes: mCYP11A1 is respectively integrated into an expression cassette with promoter 1, 3β-HSD is respectively integrated into an expression cassette with promoter 2, and the mCYP11A1 expression cassette and the 3β-HSD expression cassette are assembled into a pINA1269 integration plasmid after enzyme digestion, and finally the plasmid is linearized after NotI digestion to obtain module C;

The mCYP11A1 source includes Sus scrofa; the promoter 1 includes TEF1p; the 3β-HSD source includes Mus musculus, Bos taurus, Vaccinia virus, Arabidopsis thaliana, Mycobacterium tuberculosis or Homo sapiens (type II); the promoter 2 includes EXP1p; the assembly method includes Gibson; the enzymes used for pINA1269 digestion include SalI and ClaI.

In some specific embodiments of the present invention, the construction of the module D comprises: splicing the left arm of the IntF integration site, pUC18H, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, the right arm of the IntD integration site, CYP17A1 and the POR module to obtain a fragment containing restriction sites at both ends, and obtaining the module D after restriction digestion;

The pUC18H is pUC18H digested with HincII; the sources of the CYP17A1 and POR modules include Ovisaries and Mesocricetus auratus; the method used for splicing includes Gibson; the restriction site includes NotI; and the restriction digestion includes NotI digestion.

The present invention also provides the use of any of the following in the synthesis of steroidal compounds and/or steroidal hormone drugs:

(I), the expression element; and/or

(II), the plasmid; and/or

(III) the host.

In some specific embodiments of the present invention, the steroidal compounds and/or steroidal hormone drugs include progesterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione and/or testosterone;

In some embodiments of the invention, the progesterone is synthesized de novo in a microbial chassis using a carbon source and/or 3β-HSD.

In some specific embodiments of the present invention, the 17-hydroxyprogesterone is synthesized de novo in a microbial chassis using a carbon source, 3β-HSD, CYP17A1 and POR.

In some embodiments of the invention, the 17-hydroxypregnenolone is synthesized de novo in a microbial chassis using a carbon source, CYP17A1 and POR.

In some embodiments of the invention, the dehydroepiandrosterone is synthesized de novo in a microbial chassis using a carbon source, CYP17A1, POR and/or CYB5.

In some embodiments of the present invention, the androstenedione and testosterone are synthesized de novo in a microbial chassis using a carbon source, CYP17A1, POR, CYB5 and/or 3β-HSD.

In some specific embodiments of the present invention, the carbon source comprises glucose; the microbial chassis comprises the high-yielding campesterol Yarrowia lipolytica chassis strain SyBE_Y12060077 and/or the wild-type Yarrowia lipolytica strain ATCC201249;

The 3β-HSD source includes Mus musculus, Bos taurus, Vaccinia virus, Arabidopsisthaliana, Mycobacterium tuberculosis, Homo sapiens (type I), Homo sapiens (type II), Homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation);

The CYP17A1 and POR sources include Equus caballus, Ovis aries, Mesocricetusauratus or Xenopus laevis;

The CYB5 sources include Equus caballus, Ovis aries, Mesocricetus auratus or Susscrofa.

In some specific embodiments of the present invention, the directed synthesis of androstenedione comprises co-culturing an upstream module expressing 3β-HSD and a downstream module expressing CYP17A1, POR and/or CYB5.

In some specific embodiments of the present invention, the source of 3β-HSD includes Bos taurus; the source of CYP17A1, POR and/or CYB5 includes Mycobacterium tuberculosis and/or Ovis aries;

In some specific embodiments of the present invention, the upstream module includes the module C;

The downstream module includes one or more of the module A, the module B, the module four, the module two or the module five.

In some specific embodiments of the present invention, the construction of the upstream module includes: the mCYP11A1 is expressed under promoter 1, the 3β-HSD is expressed under promoter 2, and both are integrated into the pBR322 site at the same time; the promoter 1 includes TEF1p; the promoter 2 includes EXP1p;

The construction of the downstream module includes: the CYP17A1 and POR are both expressed under the promoter and integrated into the IntD site of the chassis strain genome; the promoter includes TEF1inp; the chassis strain includes the high-yielding campesterol Yarrowia lipolytica chassis strain SyBE_Yl2060077 and/or the wild-type Yarrowia lipolytica strain ATCC201249.

In some specific embodiments of the present invention, the combination with the highest directed synthesis of androstenedione includes co-culturing a host expressing module C with a host expressing module five, module four and/or module two; module C is selected from Bos taurus; and module two is selected from Equus caballus.

In some specific embodiments of the present invention, the synthesis of 17-hydroxyprogesterone comprises co-culturing an upstream module that co-expresses the pregnenolone pathway and/or 3β-HSD with a downstream module that only expresses CYP17A1, CYB5 and POR.

In some specific embodiments of the present invention, the source of 3β-HSD includes Bos taurus; the source of CYP17A1, POR and/or CYB5 includes Mycobacterium tuberculosis and/or Ovis aries;

The combination with the highest synthesis of 17-hydroxyprogesterone includes co-culturing a host expressing module C with a host expressing module A and/or module B; module C is selected from Bos taurus; and module A and/or module B are selected from Ovis aries.

In some specific embodiments of the present invention, the CYP17A1 that efficiently 17α-hydroxylates steroidal substrates in the directed synthesis of androstenedione and/or the synthesis of 17-hydroxyprogesterone comprises a Mesocricetus auratus source and/or an Ovisaries source.

In some specific embodiments of the present invention, the CYP17A1 with high 17,20-cleavage activity for steroidal substrates in the directed synthesis of androstenedione and/or the synthesis of 17-hydroxyprogesterone comprises a Mesocricetus auratus source and/or an Equus caballus source.

In some specific embodiments of the present invention, P5 is used as a substrate to synthesize one or more of P4, 17OHP5, 17OHP4 or DHEA.

In some specific embodiments of the present invention, synthesizing P4 using P5 as a substrate includes expressing 3β-HSD; the source of 3β-HSD includes one or more of Vaccinia virus, Bos taurus, Mycobacterium tuberculosis, Homosapiens (type I) or Homo sapiens (type II).

In some specific embodiments of the present invention, the synthesis of 17OHP5 using the P5 as a substrate comprises co-expressing CYP17A1 and POR; the sources of the CYP17A1 and POR include Ovis aries and/or Mesocricetus auratus.

In some specific embodiments of the present invention, the synthesis of 17OHP4 using P5 as a substrate includes co-expressing 3β-HSD, CYP17A1 and POR; the source of 3β-HSD includes Homo sapiens (type II) and/or Vaccinia virus; the source of CYP17A1 and POR includes Ovis aries.

In some specific embodiments of the present invention, synthesizing DHEA using P5 as a substrate comprises co-expressing CYP17A1, POR and/or CYB5; the sources of CYP17A1 and POR include Ovis aries and/or Equus caballus; and the source of CYB5 includes Equus caballus.

In some specific embodiments of the present invention, 17OHP4 is synthesized using 17OHP5 as a substrate.

In some specific embodiments of the present invention, the synthesis of 17OHP4 using 17OHP5 as a substrate includes expressing 3β-HSD; the source of 3β-HSD includes Homo sapiens (type I) and/or Homo sapiens (type II).

In some specific embodiments of the present invention, DHEA is synthesized using 17OHP5 as a substrate.

In some specific embodiments of the present invention, synthesizing DHEA using 17OHP5 as a substrate comprises co-expressing CYP17A1, POR and/or CYB5; the sources of CYP17A1 and POR include Ovis aries; the source of CYB5 includes Mesocricetus auratus, Equus caballus or Sus scrofa.

In some specific embodiments of the present invention, 4AD and TS are synthesized using DHEA as a substrate.

In some specific embodiments of the present invention, the synthesis of 17OHP4 using the 17OHP5 as a substrate includes expressing 3β-HSD; the source of the 3β-HSD includes one or more of Vaccinia virus, Mycobacterium tuberculosis, Homo sapiens (type I), Homo sapiens (type II) or Bos taurus;

In some specific embodiments of the present invention, 4AD and TS are synthesized from the 17OHP4.

In some specific embodiments of the present invention, the synthesis of 4AD and TS with 7OHP4 includes co-expressing CYP17A1, POR and/or CYB5; the sources of CYP17A1 and POR include Mesocricetus auratus, Equus caballus or Ovisaries; the sources of CYB5 include Equus caballus, Ovis aries, Mesocricetus auratus or Susscrofa.

The present invention provides a method for achieving directional synthesis of complex mesh path node path products by combining and expressing different path modules;

a) For example: directed synthesis of androstenedione (as described in Example 7);

b) For example, the synthesis of 17-hydroxyprogesterone: the upstream module bacteria: co-express the pregnenolone pathway and 3β-HSD; the downstream module bacteria: only express CYP17A1, CYB5 and POR. The upstream and downstream modules can be co-cultured to synthesize 17-hydroxyprogesterone from glucose;

Two CYP17A1s that can efficiently 17α-hydroxylate steroidal substrates in heterologous synthesis: Ma_CYP17A1 and Oa_CYP17A1;

Two CYP17A1s with efficient 17,20-cleavage activity on steroidal substrates in heterologous synthesis: Ma_CYP17A1 and Ec_CYP17A1;

Combinatorial expression of specific network pathway products in the yeast chassis achieves directed synthesis of node products;

a) Synthesis of P4 using P5 as substrate: expression of 3β-HSD;

b) Synthesis of 17OHP5 using P5 as substrate: co-expression of CYP17A1 and POR;

c) Synthesis of 17OHP4 using P5 as substrate: co-expression of 3β-HSD, CYP17A1, and POR;

d) Synthesis of DHEA using P5 as substrate: co-expression of CYP17A1, POR, and CYB5;

Combinatorial expression of homologous proteins with complementary catalytic specificity to construct efficient biotransformation synthetic pathways;

a) For the synthesis of P4 using P5 as substrate: single expression of Vv_3β-HSD;

b) For the synthesis of P4 using P5 as substrate: single expression of Bt_3β-HSD;

c) For the synthesis of P4 using P5 as substrate: single expression of Mt_3β-HSD;

d) For the synthesis of P4 using P5 as substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD);

e) For the synthesis of P4 using P5 as substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD);

f) For the synthesis of P4 using P5 as substrate: combined expression of 3β-HSD in a) to e);

g) For the synthesis of 17OHP4 using 17OHP5 as substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD);

h) For the synthesis of 17OHP4 using 17OHP5 as substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD);

i) For the synthesis of 17OHP4 using 17OHP5 as substrate: combined expression of Hs_3β-HSD1 and Hs_3β-HSD2;

j) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Vv_3β-HSD;

k) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Mt_3β-HSD;

l) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD);

m) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD);

n) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Bt_3β-HSD2;

o) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: combined expression of 3β-HSD in j) to n);

p) For DHEA synthesis using P5 as substrate: co-expression of Oa_CYP17A1, Oa_POR, Ec_CYP17A1, Ec_POR, and Ec_CYB5;

q) For the synthesis of 17OHP4 using P5 as substrate: co-expression of Hs_3β-HSD2 (type II human Hs_3β-HSD), Vv_3β-HSD, Oa_CYP17A1, and Oa_POR;

r) For the synthesis of 17OHP5 using P5 as substrate: co-expression of Oa_CYP17A1 and Oa_POR;

s) For the synthesis of 17OHP5 using P5 as substrate: co-expression of Ma_CYP17A1 and Ma_POR;

t) For the synthesis of 17OHP5 using P5 as substrate: combined expression of CYP17A1 and POR in r) to s);

u) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ma_CYB5, and Oa_POR;

v) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ec_CYB5, and Oa_POR;

w) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ss_CYB5, and Oa_POR;

x) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Ss_CYB5, Ma_POR;

y) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Ss_CYB5, and Ec_POR;

z) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Oa_CYP17A1, Ss_CYB5, and Oa_POR;

aa) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Ec_CYB5, Ma_POR;

ab) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Ec_CYB5, and Ec_POR;

ac) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Oa_CYP17A1, Ec_CYB5, and Oa_POR;

ad) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Oa_CYB5, and Ec_POR;

ae) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Ma_CYB5, and Ec_POR;

af) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Oa_CYB5, Ma_POR;

ag) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Ma_CYB5, Ma_POR.

In some specific embodiments of the present invention, the combination with the highest directed synthesis of androstenedione includes co-culturing a host expressing module C with a host expressing module five, module four and/or module two; module C is selected from Bos taurus; and module two is selected from Equus caballus.

In some specific embodiments of the present invention, in the conversion experiment with pregnenolone (P5) as the substrate, the one showing the strongest pregnenolone conversion efficiency is Vv_3β-HSD; the conversion efficiency is 6.8%; the ones showing the second strongest catalytic efficiency include type I human and mutant 3β-HSD; the catalytic efficiency is 4.6-4.8%;

In the conversion experiment with 17-hydroxypregnenolone (17OHP5) as the substrate, the ones that showed strong conversion efficiency included type I human 3β-HSD and mutant 3β-HSD, type II human 3β-HSD and mutant 3β-HSD; the type I human 3β-HSD had the highest progesterone conversion rate of 3.1%;

Taking dehydroepiandrosterone (DHEA) as a substrate, the ones showing stronger catalytic activity include type II human, bovine and mycobacterium-derived 3β-HSD; the conversion efficiency of type II human 3β-HSD is 10.5%.

In some specific embodiments of the present invention, the host comprises a

SyBE_Yl2091001~SyBE_Yl2091004 strains, SyBE_Yl2091005~SyBE_Yl2091016 and SyBE_Yl2090013~SyBE_Yl2090016 strains containing module one and module two, SyBE_Yl2091030 strain containing module five, module four and derived from Equus caballus module two, SyBE_Yl2090007 strain containing module seven, module six and derived from Vaccinia virus module three.

In some specific embodiments of the present invention, the SyBE_Yl2091001 to SyBE_Yl2091004 strains and SyBE_Yl2091005 to SyBE_Yl2091016 strains are cultured to synthesize 17-hydroxypregnenolone;

The SyBE_Yl2091030 strain is cultivated to realize efficient conversion of P5 to synthesize DHEA or 4AD, and P4 to synthesize DHEA or 4AD; in the bioconversion with P5 as the substrate, the DHEA synthesis amount of the SyBE_Yl2091030 strain reaches 12.6 mg/L, which is 7.32 times higher than that of the SyBE_Yl2091016; in the bioconversion with P4 as the substrate, the SyBE_Yl2091030 strain achieves 13.9 mg/L of androstenedione synthesis, which is 86.2 times higher than the yield of the SyBE_Yl2091016 strain;

The SyBE_Yl2090007 strain was cultured and directional synthesized 17OHP4 using P5 as a substrate; the amount of 17OHP4 synthesized reached 3.90 mg/.

In some specific embodiments of the present invention, the host further comprises an upstream module strain and a downstream module strain;

The upstream module strains include SyBE_Yl2090018, SyBE_Yl2090006, SyBE_Yl2091025 to SyBE_Yl2091028 containing the module three; the downstream module strains include SyBE_Yl2091006 containing the module A and module B and SyBE_Yl2091016 containing the module A and module B, and SyBE_Yl2091030.

In some specific embodiments of the present invention, progesterone is obtained by culturing the SyBE_Yl2090018, SyBE_Yl2090006, SyBE_Yl2091025 to SyBE_Yl2091028 strains alone.

In some specific embodiments of the present invention, the SyBE_Yl2091025 and SyBE_Yl2091006 are co-cultured to synthesize 0.25 mg/L of 17-hydroxyprogesterone, 0.74 mg/L of 17-hydroxypregnenolone, and 0.88 mg/L of androstenedione.

In some specific embodiments of the present invention, the mixed co-culture system of SyBE_Yl2091025 and SyBE_Yl2091016 synthesized 0.91 mg/L of 17-hydroxyprogesterone, 0.29 mg/L of 17-hydroxyprogesterone, and 1.03 mg/L of androstenedione.

In some specific embodiments of the present invention, the SyBE_Yl2091026-SyBE_Yl2091006 is mixed and co-cultured to synthesize 2.13 mg/L of dehydroepiandrosterone.

In some specific embodiments of the present invention, the SyBE_Yl2091025 and SyBE_Yl2091030 are co-cultured to synthesize 5.02 mg/L of androstenedione and 1.09 mg/L of testosterone with androstenedione as the main product.

In some specific embodiments of the present invention, module three in the SyBE_Yl2091025 is derived from Bostaurus; module three in the SyBE_Yl2091026 is derived from Vaccinia virus; module one and module two in the SyBE_Yl2091006 are derived from Mesocricetus auratus; module one and module two in the SyBE_Yl2091016 are derived from Ovis aries.

The present invention also provides a drug, comprising any of the following items and a pharmaceutically acceptable adjuvant or auxiliary agent:

(I), the expression element; and/or

(II), the plasmid; and/or

(III) the host.

The present invention also provides a drug combination, comprising the drug and any other effective ingredients.

The present invention also provides a method for synthesizing steroid compounds and/or steroid hormone drugs, comprising taking the host, culturing, and collecting the culture.

In some specific embodiments of the present invention, the method comprises:

(I) synthesizing progesterone de novo in a microbial chassis using a carbon source and/or 3β-HSD; and/or

(II), de novo synthesis of 17-hydroxyprogesterone in a microbial chassis using a carbon source, 3β-HSD, CYP17A1 and POR; and/or

(III) de novo synthesis of 17-hydroxypregnenolone in a microbial chassis using a carbon source, CYP17A1 and POR; and/or

(IV) de novo synthesis of dehydroepiandrosterone in a microbial chassis using a carbon source, CYP17A1, POR and/or CYB5; and/or

(V) de novo synthesis of androstenedione and testosterone in a microbial chassis using carbon sources, CYP17A1, POR, CYB5 and/or 3β-HSD; and/or

(VI) Directed synthesis of androstenedione: co-culturing an upstream module expressing 3β-HSD and a downstream module expressing CYP17A1, POR and/or CYB5; and/or

(VII) Synthesis of 17-hydroxyprogesterone: co-culturing an upstream module that co-expresses the pregnenolone pathway and/or 3β-HSD with a downstream module that only expresses CYP17A1, CYB5 and POR; and/or

(VIII) using P5 as a substrate to synthesize one or more of P4, 17OHP5, 17OHP4 or DHEA; and/or

(IX), synthesizing 17OHP4 using 17OHP5 as a substrate; and/or

(X) synthesizing DHEA using 17OHP5 as a substrate; and/or

(ⅩⅠ), synthesizing 4AD and TS using DHEA as a substrate; and/or

(ⅩII) Synthesis of 4AD and TS using 17OHP4.

In some specific embodiments of the present invention, the carbon source comprises glucose; the microbial chassis comprises the high-yielding campesterol Yarrowia lipolytica chassis strain SyBE_Y12060077 and/or the wild-type Yarrowia lipolytica strain ATCC201249;

The 3β-HSD source includes Mus musculus, Bos taurus, Vaccinia virus, Arabidopsisthaliana, Mycobacterium tuberculosis, Homo sapiens (type I), Homo sapiens (type II), Homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation);

The CYP17A1 and POR sources include Equus caballus, Ovis aries, Mesocricetusauratus or Xenopus laevis;

The CYB5 source includes Equus caballus, Ovis aries, Mesocricetus auratus or Susscrofa;

In some specific embodiments of the present invention, the source of 3β-HSD includes Bos taurus; the source of CYP17A1, POR and/or CYB5 includes Mycobacterium tuberculosis and/or Ovis aries.

In some specific embodiments of the present invention, the upstream module includes the module C;

The downstream module includes one or more of the module A, the module B, the module four, the module two or the module five.

In some specific embodiments of the present invention, the construction of the upstream module includes: the mCYP11A1 is expressed under promoter 1, the 3β-HSD is expressed under promoter 2, and both are integrated into the pBR322 site at the same time; the promoter 1 includes TEF1p; the promoter 2 includes EXP1p;

The construction of the downstream module includes: the CYP17A1 and POR are both expressed under the promoter and integrated into the IntD site of the chassis strain genome; the promoter includes TEF1inp; the chassis strain includes the high-yielding campesterol Yarrowia lipolytica chassis strain SyBE_Yl2060077 and/or the wild-type Yarrowia lipolytica strain ATCC201249.

In some specific embodiments of the present invention, the combination with the highest directed synthesis of androstenedione includes co-culturing a host expressing module C with a host expressing module five, module four and/or module two; module C is selected from Bos taurus; and module two is selected from Equus caballus.

In some specific embodiments of the present invention, the source of 3β-HSD includes Bos taurus; the source of CYP17A1, POR and/or CYB5 includes Mycobacterium tuberculosis and/or Ovis aries;

The combination with the highest synthesis of 17-hydroxyprogesterone includes co-culturing a host expressing module C with a host expressing module A and/or module B; module C is selected from Bos taurus; and module A and/or module B are selected from Ovis aries.

In some specific embodiments of the present invention, the CYP17A1 that efficiently 17α-hydroxylates steroidal substrates in the directed synthesis of androstenedione and/or the synthesis of 17-hydroxyprogesterone comprises a Mesocricetus auratus source and/or an Ovisaries source.

In some specific embodiments of the present invention, the CYP17A1 with high 17,20-cleavage activity for steroidal substrates in the directed synthesis of androstenedione and/or the synthesis of 17-hydroxyprogesterone includes Mesocricetus auratus sources and/or Equus caballus sources;

In some specific embodiments of the present invention, the synthesis of P4 using P5 as a substrate includes expressing 3β-HSD; the source of 3β-HSD includes one or more of Vaccinia virus, Bos taurus, Mycobacterium tuberculosis, Homosapiens (type I) or Homo sapiens (type II).

In some specific embodiments of the present invention, the synthesis of 17OHP5 using P5 as a substrate includes co-expressing CYP17A1 and POR; the sources of CYP17A1 and POR include Ovis aries and/or Mesocricetus auratus.

In some specific embodiments of the present invention, the synthesis of 17OHP4 using P5 as a substrate includes co-expression of 3β-HSD, CYP17A1 and POR; the source of 3β-HSD includes Homo sapiens (type II) and/or Vaccinia virus; the source of CYP17A1 and POR includes Ovis aries.

In some specific embodiments of the present invention, the synthesis of DHEA using P5 as a substrate includes co-expression of CYP17A1, POR and/or CYB5; the sources of CYP17A1 and POR include Ovis aries and/or Equus caballus; and the source of CYB5 includes Equus caballus.

In some specific embodiments of the present invention, the synthesis of 17OHP4 using 17OHP5 as a substrate includes expressing 3β-HSD; the source of 3β-HSD includes Homo sapiens (type I) and/or Homo sapiens (type II).

In some specific embodiments of the present invention, the synthesis of DHEA using 17OHP5 as a substrate includes co-expression of CYP17A1, POR and/or CYB5; the sources of CYP17A1 and POR include Ovis aries; the source of CYB5 includes Mesocricetus auratus, Equus caballus or Sus scrofa.

In some specific embodiments of the present invention, the synthesis of 17OHP4 using 17OHP5 as a substrate includes expressing 3β-HSD; the source of 3β-HSD includes one or more of Vaccinia virus, Mycobacterium tuberculosis, Homo sapiens (type I), Homo sapiens (type II) or Bos taurus.

In some specific embodiments of the present invention, the synthesis of 4AD and TS by 7OHP4 includes co-expression of CYP17A1, POR and/or CYB5; the sources of CYP17A1 and POR include Mesocricetus auratus, Equus caballus or Ovisaries; the sources of CYB5 include Equus caballus, Ovis aries, Mesocricetus auratus or Susscrofa.

The present invention provides a method for realizing the directional synthesis of complex mesh path node path products by combining and expressing different path modules:

a) For example: directed synthesis of androstenedione (as described in Example 7);

b) For example, the synthesis of 17-hydroxyprogesterone: upstream module bacteria: co-express pregnenolone pathway and 3β-HSD; downstream module bacteria: only express CYP17A1, CYB5 and POR. Mixed bacterial co-culture of upstream and downstream modules can achieve the synthesis of 17-hydroxyprogesterone from glucose;

Two CYP17A1s that can efficiently 17α-hydroxylate steroidal substrates in heterologous synthesis: Ma_CYP17A1 and Oa_CYP17A1;

Two CYP17A1s with efficient 17,20-cleavage activity on steroidal substrates in heterologous synthesis: Ma_CYP17A1 and Ec_CYP17A1;

Combinatorial expression of specific network pathway products in yeast chassis achieves directed synthesis of node products;

a) Synthesis of P4 using P5 as substrate: expression of 3β-HSD;

b) Synthesis of 17OHP5 using P5 as substrate: co-expression of CYP17A1 and POR;

c) Synthesis of 17OHP4 using P5 as substrate: co-expression of 3β-HSD, CYP17A1, and POR;

d) Synthesis of DHEA using P5 as substrate: co-expression of CYP17A1, POR, and CYB5;

Combinatorial expression of homologous proteins with complementary catalytic specificity to construct efficient biotransformation synthetic pathways;

a) For the synthesis of P4 using P5 as substrate: single expression of Vv_3β-HSD;

b) For the synthesis of P4 using P5 as substrate: single expression of Bt_3β-HSD;

c) For the synthesis of P4 using P5 as substrate: single expression of Mt_3β-HSD;

d) For the synthesis of P4 using P5 as substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD);

e) For the synthesis of P4 using P5 as substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD);

f) For the synthesis of P4 using P5 as substrate: combined expression of 3β-HSD in a) to e);

g) For the synthesis of 17OHP4 using 17OHP5 as substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD);

h) For the synthesis of 17OHP4 using 17OHP5 as substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD);

i) For the synthesis of 17OHP4 using 17OHP5 as substrate: combined expression of Hs_3β-HSD1 and Hs_3β-HSD2;

j) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Vv_3β-HSD;

k) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Mt_3β-HSD;

l) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD);

m) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD);

n) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Bt_3β-HSD2;

o) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: combined expression of 3β-HSD in j) to n);

p) For DHEA synthesis using P5 as substrate: co-expression of Oa_CYP17A1, Oa_POR, Ec_CYP17A1, Ec_POR, and Ec_CYB5;

q) For the synthesis of 17OHP4 using P5 as substrate: co-expression of Hs_3β-HSD2 (type II human Hs_3β-HSD), Vv_3β-HSD, Oa_CYP17A1, and Oa_POR;

r) For the synthesis of 17OHP5 using P5 as substrate: co-expression of Oa_CYP17A1 and Oa_POR;

s) For the synthesis of 17OHP5 using P5 as substrate: co-expression of Ma_CYP17A1 and Ma_POR;

t) For the synthesis of 17OHP5 using P5 as substrate: combined expression of CYP17A1 and POR in r) to s);

u) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ma_CYB5, and Oa_POR;

v) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ec_CYB5, and Oa_POR;

w) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ss_CYB5, and Oa_POR;

x) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Ss_CYB5, Ma_POR;

y) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Ss_CYB5, and Ec_POR;

z) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Oa_CYP17A1, Ss_CYB5, and Oa_POR;

aa) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Ec_CYB5, Ma_POR;

ab) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Ec_CYB5, and Ec_POR;

ac) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Oa_CYP17A1, Ec_CYB5, and Oa_POR;

ad) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Oa_CYB5, and Ec_POR;

ae) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Ma_CYB5, and Ec_POR;

af) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Oa_CYB5, Ma_POR;

ag) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Ma_CYB5, Ma_POR.

Specifically, the method for synthesizing steroidal compounds and/or steroidal hormone drugs comprises: culturing the host, adding a steroid substrate mother solution for incubation, and quantifying pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17OHP5), 17-hydroxyprogesterone (17OHP4), dehydroepiandrosterone (DHEA), androstenedione (4AD) or testosterone (TS);

The culturing includes culturing in a seed culture medium, a biotransformation culture medium or a YPD fermentation culture medium;

The formula of the seed culture medium includes: 20g/L glucose, 20g/L peptone, and 10g/L yeast extract powder; the culture temperature of the seed culture medium is 30°C, the rotation speed is 220rpm, and the culture time is 14 to 16h;

The formula of the biotransformation medium includes: 20g/L glucose, 20g/L peptone, and 10g/L yeast extract powder; the culture temperature of the biotransformation medium is 28°C, the rotation speed is 220rpm, and the culture time is 24h;

The formula of the YPD fermentation medium includes: 50g/L glucose, 20g/L peptone, and 10g/L yeast extract powder; the YPD fermentation medium is used for culturing at a temperature of 28°C, a rotation speed of 220rpm, and a culturing time of 8 days;

The synthesis of the steroidal compounds includes 3β-HSD isomerization conversion, 17-hydroxylation conversion, 17,20-cleavage conversion, DHEA synthesis and/or 4AD synthesis.

The steroid substrate mother liquor added to the 3β-HSD isomerization conversion includes P4 and 17OHP4 solutions; the concentration of the P4 and 17OHP4 solutions is 1.75 g/L (50% EtOH-Tween80);

The steroid substrate mother solution added in the 17-hydroxylation conversion includes P4 and P5 solutions; the concentration of the P4 and P5 solutions is 1.75 g/L (50% EtOH-Tween80);

The steroid substrate mother solution added to the 17,20-cleavage conversion includes 17OHP4 and 17OHP5 solutions; the concentration of the 17OHP4 and 17OHP5 solutions is 1.75 g/L (50% EtOH-Tween80);

The steroid substrate mother solution added to the DHEA synthesis includes a P5 solution; the concentration of the P5 solution is 3.5 g/L (50% EtOH-Tween80);

The steroid substrate mother solution added to the 4AD synthesis includes a P4 solution; the concentration of the P4 solution is 3.5 g/L (50% EtOH-Tween80);

The method for quantifying pregnenolone comprises: taking the cultured host, centrifuging, resuspending, boiling, adding saponification reaction solution to react, adding extraction solvent, concentrating, and detecting;

The centrifugal speed is 12000g, and the centrifugal time is 2min; the solution used for the resuspension is hydrochloric acid; the concentration of the hydrochloric acid is 3mol/L; the boiling temperature is 100°C; the saponification reaction solution is potassium hydroxide-methanol solution; the concentration of the potassium hydroxide-methanol solution is 2mol/L; the extraction solvent is n-hexane; the concentration is carried out by a vacuum centrifugal concentrator; the concentration temperature is 25°C, the time is 30min, and the speed is 7000rpm;

The method for quantifying progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, DHEA or androstenedione comprises: taking a cultured host, adding glass beads and an extraction solvent, concentrating, and detecting;

The extraction solvent is ethyl acetate; the concentration temperature is 25°C, the time is 1200 min, and the rotation speed is 7000 rpm.

The present invention provides directional synthesis of network pathway products. Compared with the conversion of steroid substrates by natural microbial endogenous pathways, the present invention uses the reconstructed artificial heterologous network pathway under the yeast chassis and rationally combines the pathway component proteins to achieve directional synthesis of node products. By combining and expressing homologous proteins of pathway components with different catalytic specificities, the pathway conversion efficiency can be further improved. At the same time, by constructing an artificial pregnenolone synthesis pathway under the microbial chassis and introducing specific downstream androstenedione synthesis pathway components, the directional synthesis of androstenedione pathway node compounds using simple carbon sources as substrates can be achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

FIG1 shows the synthesis pathway of androstenedione from pregnenolone in Example 1;

FIG2 shows the synthesis of progesterone from pregnenolone in Example 1;

FIG3 shows the synthesis of dehydroepiandrosterone from pregnenolone in Example 1;

FIG4 shows the synthesis of 17-hydroxyprogesterone from pregnenolone in Example 1;

FIG5 shows the synthesis of 17-hydroxypregnenolone from pregnenolone in Example 1;

FIG6 shows the isomerization efficiency of 3β-HSD from different sources in Example 1 with pregnenolone, 17-hydroxypregnenolone, and DHEA as substrates in the context of lipolytic yeast;

FIG7 shows the 17α-hydroxylation efficiency of CYP17A1 from different sources with pregnenolone and progesterone as substrates in the context of lipolytica in Example 2; wherein the left figure shows the 17α-hydroxylation efficiency of CYP17A1 from different sources with pregnenolone as substrate in the context of lipolytica; the right figure shows the 17α-hydroxylation efficiency of CYP17A1 from different sources with progesterone as substrate in the context of lipolytica;

Figure 8 shows the 17,20-cleavage efficiency of different CYP17A1 and CYB5 source combinations with 17-hydroxypregnenolone and 17-hydroxyprogesterone as substrates in the context of lipolytic yeast in Example 2; wherein the left figure shows the 17,20-cleavage efficiency of different CYP17A1 and CYB5 source combinations with 17-hydroxypregnenolone as substrate in the context of lipolytic yeast; the right figure shows the 17,20-cleavage efficiency of different CYP17A1 and CYB5 source combinations with 17-hydroxyprogesterone as substrate in the context of lipolytic yeast;

FIG9 shows the efficiency of synthesizing dehydroepiandrosterone using pregnenolone as a substrate by the recombinant strain in Example 3;

FIG. 10 shows the production of androstenedione synthesized by the recombinant strain in Example 3 using progesterone as a substrate; wherein the left side of the figure shows SyBE_Yl2091016; the right side of the figure shows SyBE_Yl2091030;

Figure 11 shows that strain SyBE_Y12090007 in Example 4 synthesizes 17-hydroxyprogesterone using pregnenolone as a substrate;

FIG12 shows the test of the effect of 3β-HSD from different sources on the synthesis of progesterone in recombinant yeast in Example 5;

FIG. 13 shows the production of androstenedione synthesized by the mixed bacteria system in Example 6 (bar graph); wherein the left side of the figure shows the production of androstenedione synthesized by the mixed bacteria SyBE_Y12091025-SyBE_Y12091016; the right side of the figure shows

The production of androstenedione synthesized by the mixed strain SyBE_Yl2091025-SyBE_Yl2091006;

Figure 14 shows the synthesis of dehydroepiandrosterone by mixed bacteria in Example 7;

FIG15 shows the efficiency of 17α-hydroxylation and 17,20-cleavage catalyzed by CYP17A1 and CYB5 components from different sources in the biotransformation test in Example 8; wherein the left figure shows the efficiency of 17α-hydroxylation catalyzed by P4 as a substrate, and the right figure shows the efficiency of 17,20-cleavage catalyzed by 17OHP4 as a substrate;

FIG. 16 shows the yield of 4AD synthesized from scratch by the mixed bacteria system in Example 8 (bar graph); wherein the left side of the figure shows the yield of 4AD synthesized by the mixed bacteria SyBE_Yl2091025-SyBE_Yl2091016; the right side of the figure shows

The yield of 4AD synthesized from scratch by the mixed bacteria SyBE_Yl2091025-SyBE_Yl2091030.

DETAILED DESCRIPTION

The present invention discloses the directional synthesis of products of a network path, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

The present invention takes the directed synthesis of androstenedione pathway node products in Yarrowia lipolytica as an example, and provides a method for decoupling the network pathway in a microbial chassis to achieve directed synthesis of target compounds. Simply put, the present invention utilizes ① the Yarrowia lipolytica chassis ② the enzyme-catalyzed substrate specificity to construct a hormone steroid synthesis pathway, and ③ according to the synthesis needs of the target compound, the pathway component proteins are selectively expressed to construct a synthesis pathway. ④ Steroid hormone compounds are synthesized from scratch using a microbial chassis. The above methods are combined and optimized to achieve efficient synthesis of specific target steroids in a microbial system.

As an unconventional yeast, Yarrowia lipolytica has the following advantages in steroid synthesis: ① The genome sequence is known, it can be genetically manipulated, and it has strong proliferation ability, which is conducive to metabolic modification and large-scale production. ② As a precursor for the synthesis of steroids, the metabolic flux of acetyl-CoA is high in Y. lipolytica, which is conducive to steroid synthesis. ③ After metabolic modification, the expression of heterologous proteins in this species is high. At the same time, compared with Saccharomyces cerevisiae, the post-transcriptional glycosylation modification of the cell is closer to that of mammalian cells, which is conducive to the expression of mammalian proteins and the synthesis of animal-derived steroids. ④ With GRAS (generally regarded as safe) level of safety, it can be considered as a chassis for drug synthesis. ⑤ Yarrowia lipolytica has a wide spectrum of substrates. In addition to glucose, it can also use oils as substrates, so it can use industrial by-products and waste to produce target products. ⑥ When Y. lipolytica uses oil as a carbon source, the accumulation of intracellular lipids will cause the lipid droplets to become larger, providing storage space for the storage of non-polar products (such as steroids), which is conducive to reducing the burden of product accumulation on cells. ⑦ Compared with Saccharomyces cerevisiae, the traditional host for heterologous steroid synthesis, Saccharomyces lipolytica does not have the homologous gene of ATF2 (alcohol O-acetyltransferase), which causes steroid esterification in Saccharomyces cerevisiae and hinders further biotransformation of steroids in cells.

In the present invention, under the yeast chassis with a relatively pure and clear metabolic background, the exogenous steroid synthesis pathway is orthogonal to the endogenous metabolism of the microorganism, so that the steroid biotransformation can be carried out in a relatively pure background, and the directional synthesis of the intermediate node product can be better achieved.

The exploration of de novo synthesis of steroid hormone compounds using microorganisms is relatively limited. The present invention realizes the directional synthesis of androstenedione network pathway node products using simple carbon sources.

The construction of artificial synthetic pathways can achieve the directed synthesis of pathway node compounds and pathway flux enhancement through the combination and heterologous expression of different catalytic specific proteins.

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for achieving directed synthesis of a target compound.

The present invention provides the following:

1. Construct artificial pathways in the microbial chassis to achieve steroid synthesis.

2. Construct an artificial pathway in the Yarrowia lipolytica chassis to achieve steroidal compound synthesis.

3. A method for synthesizing 17-hydroxyprogesterone from scratch in a microbial chassis by using the combined expression of nine 3β-HSDs and four CYP17A1s in Example 1.

4. Methods for synthesizing 17-hydroxypregnenolone de novo in a microbial chassis using the four CYP17A1 in the table of Example 1 using combination expression.

5. A method for synthesizing dehydroepiandrosterone from scratch in a microbial chassis by using the combined expression of the four CYP17A1 and four CYB5 in the table of Example 1.

6. A method for synthesizing androstenedione and testosterone from scratch in a microbial chassis by using the combined expression of the table in Example 1: 4 CYP17A1, 4 CYB5, and 9 3β-HSD.

7. A method for synthesizing 17-hydroxyprogesterone from scratch in the Yarrowia lipolytica chassis using the combined expression of the nine 3β-HSDs and four CYP17A1s in the table of Example 1.

8. A method for synthesizing 17-hydroxypregnenolone de novo in the Yarrowia lipolytica chassis using the four CYP17A1 in the table of Example 1 using combined expression.

9. A method for synthesizing dehydroepiandrosterone from scratch in the Yarrowia lipolytica chassis by using the combined expression of the four CYP17A1 and four CYB5 in the table of Example 1.

10. A method for synthesizing androstenedione and testosterone from scratch in the Yarrowia lipolytica chassis by using the combined expression of 4 CYP17A1, 4 CYB5, and 9 3β-HSD in the table of Example 1.

11. Using bovine 3β-HSD, a method was developed to construct an engineered bacterium that can synthesize progesterone from scratch using a simple carbon source.

12. A method for constructing an engineered bacterium that can synthesize progesterone from scratch using human 3β-HSD.

13. A method for constructing an engineered bacterium that can synthesize progesterone from scratch using a simple carbon source by using 3β-HSD derived from vaccinia virus.

14. A method for constructing an engineered bacterium that can synthesize progesterone from scratch using a simple carbon source by using mycobacterium-derived 3β-HSD.

15. A method for achieving directed synthesis of complex mesh path node products by combining different path modules.

a) For example: Directed synthesis of androstenedione (as described in Example 7)

b) For example, the synthesis of 17-hydroxyprogesterone: the upstream module bacteria: co-express the pregnenolone pathway and 3β-HSD; the downstream module bacteria: only express CYP17A1, CYB5 and POR. The upstream module and the downstream module can be co-cultured to synthesize 17-hydroxyprogesterone from glucose.

16. Two CYP17A1s that can efficiently 17α-hydroxylate steroidal substrates in heterologous synthesis: Ma_CYP17A1 and Oa_CYP17A1

17. Two CYP17A1s with high 17,20-cleavage activity for steroidal substrates in heterologous synthesis: Ma_CYP17A1 and Ec_CYP17A1

18. Combinatorial expression of specific network pathway products in the yeast chassis enables directed synthesis of node products.

a) Synthesis of P4 using P5 as substrate: Expression of 3β-HSD

b) Synthesis of 17OHP5 using P5 as substrate: co-expression of CYP17A1 and POR

c) Synthesis of 17OHP4 using P5 as substrate: co-expression of 3β-HSD, CYP17A1, and POR

d) Synthesis of DHEA using P5 as substrate: Co-expression of CYP17A1, POR, and CYB5

19. Combined expression of homologous proteins with complementary catalytic specificity to construct efficient biotransformation synthesis pathways

a) For the synthesis of P4 using P5 as substrate: single expression of Vv_3β-HSD

b) For the synthesis of P4 using P5 as substrate: single expression of Bt_3β-HSD

c) For the synthesis of P4 using P5 as substrate: single expression of Mt_3β-HSD

d) For the synthesis of P4 using P5 as substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD)

e) For the synthesis of P4 using P5 as substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD)

f) For the synthesis of P4 using P5 as substrate: Combined expression of 3β-HSD in a) to e)

g) For the synthesis of 17OHP4 using 17OHP5 as a substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD)

h) For the synthesis of 17OHP4 using 17OHP5 as a substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD)

i) For the synthesis of 17OHP4 using 17OHP5 as a substrate: combined expression of Hs_3β-HSD1 and Hs_3β-HSD2

j) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Vv_3β-HSD

k) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Mt_3β-HSD

l) For the synthesis of 4AD and TS (testosterone) using DHEA as a substrate: single expression of Hs_3β-HSD1 (type I human Hs_3β-HSD)

m) For the synthesis of 4AD and TS (testosterone) using DHEA as a substrate: single expression of Hs_3β-HSD2 (type II human Hs_3β-HSD)

n) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: single expression of Bt_3β-HSD2

o) For the synthesis of 4AD and TS (testosterone) using DHEA as substrate: Combined expression of 3β-HSD in j) to n)

p) For DHEA synthesis using P5 as substrate: co-expression of Oa_CYP17A1, Oa_POR, Ec_CYP17A1, Ec_POR, Ec_CYB5

q) For the synthesis of 17OHP4 using P5 as substrate: co-expression of Hs_3β-HSD2 (type II human Hs_3β-HSD), Vv_3β-HSD, Oa_CYP17A1, Oa_POR

r) For the synthesis of 17OHP5 using P5 as substrate: co-expression of Oa_CYP17A1, Oa_POR

s) For the synthesis of 17OHP5 using P5 as substrate: co-expression of Ma_CYP17A1, Ma_POR

t) For the synthesis of 17OHP5 using P5 as substrate: Combined expression of CYP17A1 and POR in r) to s)

u) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ma_CYB5, Oa_POR

v) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ec_CYB5, Oa_POR

w) For DHEA synthesis by 17OHP5: co-expression of Oa_CYP17A1, Ss_CYB5, Oa_POR

x) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Ss_CYB5, Ma_POR

y) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ec_CYP17A1, Ss_CYB5, Ec_POR

z) For the synthesis of 4AD and TS (testosterone) from 17OHP4: co-expression of Oa_CYP17A1, Ss_CYB5, Oa_PORaa) For the synthesis of 4AD and TS (testosterone) from 17OHP4: co-expression of Ma_CYP17A1, Ec_CYB5, Ma_PO

ab) For the synthesis of 4AD and TS (testosterone) with 17OHP4: co-express Ec_CYP17A1, Ec_CYB5, and Ec_PORac) For the synthesis of 4AD and TS (testosterone) with 17OHP4: co-express Oa_CYP17A1, Ec_CYB5, and Oa_PORad) For the synthesis of 4AD and TS (testosterone) with 17OHP4: co-express Ec_CYP17A1, Oa_CYB5, and Ec_PORae) For the synthesis of 4AD and TS (testosterone) with 17OHP4: co-express Ec_CYP17A1, Ma_CYB5, and Ec_PORaf) For the synthesis of 4AD and TS (testosterone) with 17OHP4: co-express Ma_CYP17A1, Oa_CYB5, and Ma_POR

ag) For the synthesis of 4AD and TS (testosterone) by 17OHP4: co-expression of Ma_CYP17A1, Ma_CYB5, Ma_POR

The recombinant strains and plasmids involved in the present invention are shown in Table 1 and Table 2:

Table 1: Recombinant strains involved in the present invention

Table 2: Plasmids involved in the present invention

The optimized sequence involved in the present invention is:

Ss_mCYP11A1 (SEQ NO: 5):

ATGATCTCTACCAAGACCCCCCGACCCTTCTCTGAGATCCCCTCCCCCGGAGACAAC

GGTTGGATTAACCTGTACCGATTCTGGAAGGAGAAGGGAACCCAGAAGATCCACTA

CCACCACGTGCAGAACTTCCAGAAGTACGGCCCCATCTACCGAGAGAAGCTGGGAA

ACCTGGAGTCCGTCTACATCATTGACCCCGAGGACGTGGCCCTGCTGTTCAAGTTCG

AGGGCCCCAACCCCGAGCGATACAACATTCCCCCCTGGGTCGCCTACCACCAGCACT

ACCAGAAGCCCGTGGGTGTCCTGCTGAAGAAGTCTGGCGCTTGGAAGAAGGACCGA

CTGGTCCTGAACACCGAGGTCATGGCCCCCGAGGCTATCAAGAACTTCATTCCCCTG

CTGGACACCGTGTCCCAGGACTTCGTGGGCGTCCTGCACCGACGAATCAAGCAGCA

GGGTTCTGGCAAGTTCTCCGGAGACATTCGAGAGGACCTGTTCCGATTCGCCTTCGA

GTCTATCACCAACGTCATTTTCGGCGAGCGACTGGGAATGCTGGAGGAGATCGTGG

ACCCCGAGGCCCAGAAGTTCATTGACGCTGTCTACCAGATGTTCCAACCTCCGTGC

CTATGCTGAACCTGCCTCCCGACCTGTTCCGACTGTTCCGAACCAAGACCTGGCGAG

ATCACGTCGCCGCTTGGGACACCATCTTCAACAAGGCCGAGAAGTACACCCAGAAC

TTCTACTGGGACCTGCGACGAAAGCGAGAGTTCAACAACTACCCCGGAATTCTGTAC

CGACTGCTGGGTAACGACAAGCTGCTGTCTGAGGACGTCAAGGCCAACGTGACCGA

GATGCTGGCTGGCGGAGTGGACACCACCTCTATGACCCTGCAGTGGCACCTGTACG

AGATGGCCCGATCCCTGAACGTCCAGGAGATGCTGCGAGAGGAGGTGCTGAACGCC

CGACGACAGGCTCAGGGAGACACCTCCAAGATGCTGCAGCTGGTCCCCCTGCTGAA

GGCTTCTATCAAGGAGACTCTGCGACTGACCCCATTTCCGTGACCCTGCAGCGATA

CCTGGTCAACGACCTGGTGCTGCGAGACTACATGATCCCTGCTAAGACCCTGGTGCA

GGTCGCTGTGTACGCTATGGGTCGAGATCCCGCTTTCTTCTCTAACCCCGGACAGTT

CGACCCTACCCGATGGCTGGGCAAGGAGCGGGACCTGATCCACTTCCGAAACCTGG

GATTCGGTTGGGGCGTCCGACAGTGCGTCGGACGACGAATTGCCGAGCTGGAGATG

ACCCTGTTCCTGATCCACATTCTGGAGAACTTCAAGGTCGAGCTGCAGCACTTCTCC

GACGTGGACACCATCTTCAACCTGATTCTGATGCCCGACAAGCCCATTTTCCTGGTG

TTCCGACCCTTCAACCAGGACCCCCTGCAGGCTTAA

Oa_CYP17A1 (SEQ NO: 6):

ATGTGGGTCCTGCTGGCCGTCTTCCTGCTGACCCTGGCCTACCTGTTCTGGCCCAAG

ACCAAGCACTCCGGCGCCAAGTACCCCCGATCCCTGCCCTCCCTGCCCCTGGTTGGC

TCCCTGCCTTTCCTCCCGACGAGGCCAGCAGCACGAGAACTTCTTCAAGCTGCAG

GAGAAGTACGGCCCCATCTACTCCTTCCGACTGGGCTCCAAGACCACCGTCATGATT

GGTCACCACCAGCTGGCCCGAGAGGTCCTGCTCAAGAAGGGTAAGGAGTTTCCGG

CCGACCCAAGGTCGCTACCCTGGACATCCTGTCCGACAACCAGAAGGGCATCGCCTT

CGCTGACCACGGCGCCCACTGGCAGCTGCACCGAAAGCTGGTCCTCAACGCCTTTTGC

CCTGTTCAAGGACGGCAACCTGAAGCTGGAGAAGATCATTAACCAGGAGGCTAACG

TCCTGTGCGACTTCCTGGCCACCCAGCACGGCCAGTCCATCGACCTCTCCGAGCCCC

TGTCCCTCGCCGTGACCAACATCATTTCCTTCATTTGCTTCAACTTCTCCTTTAAGAA

CGAGGACCCCGCCCTCAAGGCCATCCAGAACGTTAACGACGGCATTCTCGAGGTGC

TCGGCAAGGAGGTGCTGCTGGACATCTTCCCTGCCCTGAAGATCTTCCCCTCCAAGG

CCATGGAGAAGATGAAGGGCTGCGTGGAGACTCGAAACGAGCTGCTGTCCGAGATT

CTGGAGAAGTGCCAGGAGAACTTCACCTCCGACTCTATTACCAACCTGCTGCACATC

CTGATGCAGGCCAAGGTCAACGCTGACAACAACACCGGCCCCGAGCAGGACTC

CAAGCTGCTGTCCAACCGACACATGCTCGCCACCATCGCCGACATCTTCGGCGCCGG

CGTCGAGACTACCACCTCCGTCATCAAGTGGATCGTCGCCTACCTGCTGCACCACCC

CTCCCTGAAGAAGCGAATCCAGGACTCCATCGACCAGAACATCGGATTCAACCGAA

CCCCCACCATCTCCGACCGAAACCGACTGGTCCTGCTCGAGGCCACCATCCGAGAG

GTCCTCCGAATCCGACCCGTCGCCCCCATGCTCATCCCCCACAAGGCCATCATCGAC

TCCTCCATCGGCGACCTGACCATCGACAAGGGCACCGACGTCGTCGTCAACCTGTGG

GCCCTGCACCACAACGAGAAGGAGTGGCAGCAGCCCGACCTCTTCATGCCCGAGCG

ATTTCTGGACCCCACCGGAACCCAGCTGATCTCCCCCTCCCTGTCCTACCTCCCCTTC

GGCGCCGGTCCCCGATCCTGTGTCGGCGAGATGCTCGCCCGACAGGAGCTGTTTCTG

TTTATGTCTCGACTCCTGCAGCGATTCAACCTGGAGATCCCCGACGACGGCAAGCTC

CCCTCCCTGGAGGGCAACCCCTCCCTGGTTCTGCAGATCAAGCCCTTCAAGGTCAAG

ATCGAGGTCCGACAGGCCTGGAAGGAGGCCCAGGCCGAGGGTTCTACCTCCTAA

Ma_CYP17A1 (SEQ NO: 7):

ATGTGGGAGCTGGTCGCCCTGCTGCTGCTGACCCTGGCCTACTTCTTCTGGTCCAAG

TCCAAGACCTGCGGCGCCAAGTCCCCCAAGTCCCTGCCCTTCCTGCCCCTGGTCGGC

TCCCTGCCTTTATCCCCCGACACGGCCACCCCCACGTCAACTTCTTCAAGCTGCAG

GAGAAGTACGGCCCCATCTACTCCCTCCGACTGGGCTCCACCACCACCGTCATCATT

GGCCAGTACCAGCTCGCCAAGGAGGTCCTGGTCAAGAAGGGTAAGGAGTTCTCCGG

CCGACCCCACATGGTTACCCTGGGCCTGCTGTCCGACCAGGGCAAGGGCATCGCTTT

CGCCGACTCCGGCGGATCTTGGCAGCTGCACCGAAAGCTGGCCCTCTCCTCCTTTGC

TCTGTTCCGAGATGGTAACCAGAAGCTGGAGAAGATCATCTGCCAGAAGGCTTCCTC

CCTGTGTGACTTTCTGCTGACCCACAACGAGGAGTCCATCGACCTGTCCGAGCCCAT

CTTCAACTCCATCACCAACATCATCTGCATCATTTGCTTCGGCATCTCCTACGAGAAC

CGAGATCCTATCCTCGCCACCATCAAGTCCTTTACCGAGGGCATTCTGAACTCCCTC

GGCAACGACCACCTCGTCGATATCTTCCCCTGGCTGACCATCTTCCCCCAACAAGACC

GTCGATATGATCAAGAAGAACGTCAAGATCCGAGATGAAGTGCTGTCCGGCATCCT

GGAGAAGTGCAAGGAGAAGTTTAACTCCGACTCCATCTCCTCCCTGATGGACCTGCT

GATCCAGGCCAAGACCAACGCCGACAACAACACCTCCGAGGGCCAGGGCTCCA

ACGCCTTCTCCGACATGCACATCCTGGCCACCATCGCCGACATCTTCGGCGCCGGAA

TCGAGACTACCGCCTCCGTCCTGTCTTGGATCATCGCCTTCCTCCTGCACAACCCCGA

GGTCAAGAAGAAGATCCAGAAGGAGATTGACCAGAACATCGGATTCTCCCGAACCC

CCACCTTCAACGACCGAAACCACCTGCTGATGCTGGAGGCCACCATCCGAGAGGTC

CTGCGAATCCGACCCGTCGCCCCCATGCTGATCCCCCACCGAGCCAACTCCGACATG

TCCATCGGCGAGTTCTCCATCCCCAAGTTCACCCCCGTCATCATCAACCTGTGGGCC

CTGCACCACTCCGAGAAGGAGTGGGACCAGCCCGACCGATTCATGCCCGAGCGATT

TCTGGACCCCACCGGATCTCACCTCATCACCCCCTCCCTCTCCTACCTGCCCTTCGGC

GCCGGCGCTCGATCCTGTATCGGCGAGGTCCTCGCCCGACAGGAGCTGTTCCTGTTT

ATGGCCCACCTCCTGCAGCGATTCGACCTCGACGTCCCCGACGACGAGCAGCCCCCT

TGCCTGAAGGGTAACGCCAACGTCGTGTTTCTGATCGACCCCTTCAAGGTCAAGATT

ACCGTCCGACAGGCCTGGAAGGACGCCCAGGCCGAGGTTAACACCTGGCGACCCTA

A

Ec_CYP17A1 (SEQ NO: 8):

ATGTGGGAGCTGCTGGCCTTCCTGCTGCTGGCCATCGCCTACTTCTTCCGACCCAAG

GTCAAGTGCCCCGGCGCCAAGTACCCCAAGTCCCTGCCCTACCTGCCCCTGGTCGGC

TCCCTGCCTTTCCTCCCGACACGGCCACCCCCACGTCAACTTCTTCAAGCTCCAGA

AGAAGTACGGTCCTATCTACTCCCTGCGAATGGGCACCAAGACCACCGTCATGGTCG

GTCACTACCAGCTGGCCAAGGAGGTCCTGATCAAGAAGGGTAAGGAGTTCTCCGGC

CGACCCCAGGTCGCCACCCTGAACATCCTCTCCGACAACCAGAAGGGCGTCGCCTTC

GCTGACCACGGCGCCCCTTGGCAGCTGCACCGAAAGCTGGTCCGAGCCGCCTTCGCC

CTGTTCAAGGACGGCAACCAGAAGCTGGAGAAGATCATTTGCCACGAGACTTCCCT

GCTGTGCGACCTCCTGGCCACCCAGAACGGCCAGACCATCGACCTGTCCTCCCCCCT

CTTCCTGGCCGTGACCAACGTCATCTGCTGGATCTGCTTCAACTCCTCCTACATGAA

GGGCGACCCCGCCCTCGAGACTATGCAGAACTACCACAAGGGCATTCTCGAGACTC

TCGAGAAGGACAACGTCGTCGATATTTTCCCCGCCCTGAAGATCTTCCCCAACAAGT

CCCTGGAGAAGATGCGACACTGTGTGAACATCCGAAACGAGCTGCTGTCCAAGATC

TTCGAGAAGCACAAGGAGAACTTTAACTCCGACTCCATCACCTCCATGCTCGACCTC

CTGATCCAGGCCAAGAAGAACTCCGACAACAACAACACCGGCCCCGACCAGGACTC

CAAGCTCCTGTCCGACAAGCACATCCTCGCCACCATCGGCGACATCTTCGGCGCCGG

CGTCGAGACTACCACCTCCGTCGTCAAGTGGATCGTCGCCTTCCTGCTCCACGACCC

CCAGCTGAAGAAGAAGATCCAGGAGGAGATCGACCAGAACGTCGGATTCTCCCGAA

CCCCCACCCTGTCCGACCGAAACCGACTGCTGCTGCTGGAGGCCACCATCCGAGAG

GTTCTCCGAATCCGACCCGTCGCCCCCATGCTGATCCCCCACAAGGCCCTCGTCGAT

TCCTCCATCGGCGAGTTCGCCGTCGATGACGGCACCAACGTCATTATTAACCTGTGG

GCCCTGCACCACAACGAGAAGGAGTGGCACCAGCCCGACCGATTCATGCCCGAGCG

ATTTCTGGACCCCACCGGCTCCCAGCTGATCTCCCCCTCCCTGTCCTACCTGCCCTTC

GGCGCCGGTCCCCGATCCTGTATCGGCGAGCTCCTCGCCCGACAGGAGCTGTTTCTG

TTCACCGCCTGGCTCCTGCAGCGATTCAACCTGGAGGTCCCCGACGACGGCAGCTC

CCTTCCCTGGAGGGCCACCCTACCGCCGTCTTTCTGATTGACTCCTTCAAGGTCAAG

ATTAACGTCCGACAGGCCTGGCGAGAGGCTCAGGCCGAGGGTTCACCTAA

Xl_CYP17A1 (SEQ NO: 9):

ATGATCTCCTACGTCGCCGGCGCCCTGCTGCTGGCTTTCGGTCTGGCCCTGATCTCCG

TCTGGAAGTTCGCTGGTGGCAAGCACCGAGGCGCTAAGTACCCCAACTCCCTGCCCT

GCCTGCCCTTCATCGGTTCCCTGCTGCACATCGGCAACCACCTGCCCCCCCACATCCT

GTTTTGCAAGCTCCAGGAGAAGTACGGCTCCCTGTACTCCTTCCGAATGGGCTCCCA

CTACATCGTCATCGTCAACCACCACGAGCACGCCAAGGAGGTCCTCCTCAAGAAGG

GCAAGACCTTTGGCGGCCGAACCCCGAGCCGTTACCACCGACATCCTCACCCGAAAC

GCCAAGGACATCGCCTTCGCCAACTACTCCCCCTCCTGGAAGTTCCACCGAAAGGTC

GTCCACGCCGCCCTCTCCATGTTTGGCGAGGGTACTGTCGCCATCGAGAAGATCATC

TCCCGAGAGGCCACCTCCCTGTGCCAGTCCCTCATCTCCTTTCCAGGACAACCCCCTG

GACATGGCCCCCGAGCTCACCCGAGCCGTTACCAACGTCGTTCTGCGCCCTGTGCTTC

AACACCCGATACAAGCGATGCGACCCCGAGTTCGAGGAGATGCTGGCCTACTCCAA

GGGCATCGTCGATACCGTGGCCAAGGACTCTCTGGTCGATATTTTCCCCTGGCTGCA

GATCTTCCCCAACAAGGACCTGGACATTCTGAAGCGATCCGTGGCCATCCGAGACA

AGCTGCTGCAGAAGAAGCTCAAGGAGCACAAGGAGGCTTTCTGCAACGAGGAGGTT

AACGACCTGCTGGACGCCCTGCTGAAGGCCAAGCTGTCCATGGAGAACAACAACTC

CAACATCTCCCAGGAGGTCGGCCTCACCGACGACCACCTGCTGATGACCGTCGGCG

ACATTTTCGTCGCCGGCGTGGAGACTACCACCACCGTCCTGAAGTGGACCATGGCCT

ACCTCCTGCACTACCCCGAGGTCCAGACCAAGATTCAGGAGGAGCTGGACTTCAAG

GTTGGCTTCGGCCGACACCCCGTCCTGTCCGACCGACGAATTCTGCCCTACCTCGAC

GCCACCATCTCCGAGGTCCTCCGAATCCGACCCGTCGCCCCCCTGCTGATCCCCCAC

GTTGCCCTGCAGGAGTCCTCCATCGCCGAGTACACCATCCCCCAGGACGCCCGAGTC

GTGATTAACCTGTGGTCCCTGCACCACGACCCCAACGAGTGGGAGAACCCCGAGGA

GTTCAACCCCGAGCGATTTCTCGACGAGAACGGAAACCACGTCTACTCCCCCTCTCA

GTCTTACCTGCCCTTTCGGCGCCGGCATCCGAGTCTGCCTGGGCGAGGCTCTGGCCAA

GATGGAGGTCTTTCTGTTCCTGTCCTGGATTCTGCAGCGATTCACCCTGGAGCTGCCC

GCCGGCGACTCTCTCCCTGACCTGGACGGCAAGTTTTGGCGTGGTTCTGCAGGTCAAG

AAGTTTCGAGTTACCACCAAGCTGCGAGAGGCCTGGAAGAACATCGACCTCACCAC

CTAA

Oa_POR (SEQ NO: 10):

ATGAACATGGGCGACTCCAACATGGACGCTGGCACCACCACCCCCGAGACTGTGGC

CGAGGAGGTCAGCTTATTTTCCACCACCGACATGATCCTGTTTCCCTGATCGTCGG

CGTCATGACCTACTGGTTCCTCTCCGAAAGAAGAAGGAGGAGGTCCCCGAGTTCAC

CAAGATTCAGACCACCACCTCCTCCGTCAAGGACCGATCCTTCGTGGAGAAGATGA

AGAAGACCGGCCGAAACATCATCGTCTTTTACGGCTCCCAGACCGGTACTGCCGAG

GAGTTCGCCAACCGACTCTCCAAGGACGCCCACCGATACGGCATGCGAGGCATGGC

CGCCGACCCCGAGGAGTACGACCTCGCTGACCTCTCCTCCCTCCCCGAGATCGAGAA

GGCCCTGGGCTGTTTTCTGCATGGCCACCTACGGTGAGGGCGACCCCACCGACAACGC

CCAGGACTTTTACGACTGGCTGCAGGAGACTGACGTCGATCTCTCCGGCGTCAAGTA

CGCCGTTTTCGCCCTGGGTAACAAGACCTACGAGCACTTCAACGCTATGGGCAAGTA

CGTCGATAAGCGACTGGAGCAGCTGGGCGCCCAGCGAATTTTTGACCTGGGTCTGG

GCGACGACGACGGCAACCTGGAGGAGGACTTCATCACCTGGCGAGAGCAGTTCTGG

CCCGCCGTCTGCGAGCACTTTGGAGTCGAGGCTACCGGCGAGGAGTCTTCCATTCGA

CAGTACGAGCTGATGGTGCACACCGACATGGACATGGCCAAGGTCTACACCGGCGA

GATGGGCCGACTGAAGTCCTACGAGAACCAGAAGCCCCCCTTCGACGCCAAGAACC

CCTTCCTGGCCGTGGTCACCACCAACCGAAAGCTGAACCAGGGTACTGAGCGACAC

CTGATGCACCTGGAGCTGGACATCTCCGACTCCAAGATCCGATACGAGTCCGGCGA

CCACGTCGCCGTCTACCCTGCCAACGACTCCGCCCTGGTCAACCAGCTGGGCGAGAT

CCCTCGGCGCCGACCTCGACGTCATCATGTCCCTGAACAACCTCGACGAGGAGTCCAA

CAAGAAGCACCCCTTCCCCTGCCCCACCTCCTACCGAACCGCCCTGACCTACTACCT

GGACATCACCAACCCCCCCCGAACCAACGTCCTCTACGAGCTGGCCCAGTACGCCTC

CGAGCCCGCTGAGCAGGAGCAGCTCCGAAAGATGGCCTCCTCCTCCGGCGAGGGCA

AGGAGCTGTACCTCCGATGGGTCCTGGAGGCCCGACGACACATTCTCGCCATTCTGC

AGGACTACCCCTCCCTGCGACCCCCCATCGACCACCTGTGCGAGCTCCTGCCCCGAC

TCCAGGCCCGATACTACTCCATTGCCTCTTCTCCAAGGTTCACCCCAACTCCGTTCA

CATCTGCGCCGTCGCCGTCGAGTACGAGACTAAGACCGGTCGAATCAACAAGGGCG

TCGCCACCTCCTGGCTGCGAGCCAAGGAGCCCGCCTCCGAGAACGGCGGTCGAGCT

CTGGTCCCCCATGTACGTGCGAAAGTCCCAGTTCCGACTGCCCTTCAAGGCCACCACC

CCCGTCATCATGGTTGGCCCCGGCACCGGCGTGGCCCCTTTTATCGGCTTCATCCAG

GAGCGAGCCTGGCTGCGACAGCAGGGCAAGGAGGTTGGCGAGACTCTCCTGTACTA

CGGCTGCCGACGATCCGACGAGGACTACCTGTACCGAGAGGAGCTGGCCGGCTTCC

ACAAGGACGGCACCCTGACCCAGCTCAACGTTGCCTTCTCTCGAGAGCAGCCCCAG

AAGGTCTACGTCCAGCACCTGCTCAAGAAGGACAAGGAGCACCTGTGGAAGCTGAT

TCACGAGGGAGGCTCACATCTACGTCTGCGGCGACGCCCGAAACATGGCCCGAG

ATGTTCAGAACACCTTCTACGACATCGTCGCCGAGCAGGGAGCCATGGAGCAGGCC

CAGGCCGTCGATTACGTGAAGAAGCTGATGACCAAGGGACGATACTCCCTGGACGT

CTGGTCCTAA

Ma_POR (SEQ NO: 11):

ATGACCGAGGCCGTGGCCGAGGAGGTCAGCTTATTTTCCACCACCGACGTCGTCCTG

TTCTCCCTGATCGTCGGCGTCCTGACCTACTGGTTCATCTTCCGAAAGAAGAAGGAG

GAGGTCCCCGAGTTTTCTAAGATTCAGACCGCCACCCCCTCCGTCAAGGAGTCCTCT

TTCGTTGAGAAGATGAAGAAGACCGGCCGAAACATCATCGTGTTCTACGGATCTCA

GACCGGTACTGCCGAGGAGTTTGCCAACCGACTCTCCAAGGACGCCCACCGATACG

GCATGCGAGGCATGTCCGCCGACCCCGAGGAGTACGACCTCGCCGACCTCTCCTCTC

TGCCCGAGATTGACAAGTCCCTGGGTCGTTTTCTGCATGGCCACCTACGGAGAGGGCG

ACCCCACCGACAACGCCCAGGACTTCTACGACTGGCTGCAGGAGACTGACGTCGAT

CTGACCGGCGTGAAGTTCGCCGTCTTCGGCCTGGGCAACAAGACCTACGAGCACTTT

AACGCCATGGGCAAGTACGTCGATCAGCGACTGGAGCAGCTGGGCGCCCAGCGAAT

CTTCGAGCTGGGTCTGGGCGACGACGACGGCAACCTCGAGGAGGACTTCATCACCT

GGCGAGAGCAGTTCTGGCCCGCCGTCTGCGAGTTCTTTGGAGTCGAGGCTACCGGCG

AGGAGTCTTCCATCCGACAGTACGAGCTGCTGGTCCACGAGGACATCGACGCCGCC

AAGGTCTACACCGGCGAGATGGGCCGACTGAAGTCCTACGAGAACCAGAAGCCCCC

CTTTGACGCCAAGAACCCCTTCCTCCGCCGCCGTCACCACCAACCGAAAGCTGAACCA

GGGTACTGAGCGACACCTGATGCACCTGGAGCTGGACATCTCCGACTCCAAGATCC

GATACGAGTCCGGTGACCACGTCGCCGTCTACCCCGCCAACGACTCCACCCTGGTCA

ACCAGATCGGCGAGATTCTCGGCGCCGACCTCGACGTCGTGATGTCCCTGAACAACC

TGGACGAGGAGTCCAACAAGAAGCACCCCTTCCCCTGCCCCACCACCTACCGAACC

GCTCTCACCTACTACCTGGACATCACCAACCCCCCCCGAACCAACGTCCTCTACGAG

CTGGCCCAGTACGCCTCCGAGCCCTCCGAGCAGGAGCAGCTCCACAAGATGGCCTC

CTCCTCCGGCGAGGGCAAGGAGCTGTACCTCTCCTGGGTCGTCGAGGCCCGACGAC

ACATTCTCGCCATCCTCCAGGACTACCCCTCCCTGCGACCCCCCATCGACCACCTGT

GTGAGCTCCTCCCTCGACTGCAGGCCCGATACTACTCCATCGCCTCCTCCTCTAAGG

TCCACCCCAACTCCGTCCACATCTGCGCCGTCGCCGTCGAGTACGAGGCCAAGTCCG

GCCGAGTGAACAAGGGCGTTGCCACCTCCTGGCTGCGAGCCAAGGAGCCCGCCGGT

GAGAACGGCCGACGAGCTCTGGTCCCCATGTTTGTCCGAAAGTCCCAGTTTCGACTG

CCCTTCAAGTCTGTCACCCCCGTTATTATGGTTGGTCCCGGCACCGGCATTGCCCCCT

TCATGGGCTTCATTCAGGAGCGAGCCTGGCTGCGAGAGCAGGGCAAGGAGGTCGGC

GAGACTCTCCTGTACTACGGCTGCCGACGATCCGACGAGGACTACCTGTACCGAGA

GGAGCTGGCCCGATTCCACAAGGACGGTGCCCTGACCCAGCTGAACGTGGCCTTTTC

CCGAGAGCAGGCCCACAAGGTCTACGTCCAGCACCTGCTGAAGCGAGACAGAGAGC

ACCTGTGGAAGCTGATCCACGAGGGCGGAGCTCACATCTACGTCTGCGGCGACGCC

CGAAACATGGCCAAGGACGTCCAGAACACCTTCTACGACATTGTCGCCGAGTTTGG

CCCCATGGAGCACGCCCAGGCCGTCGATTACGTGAAGAAGCTGATGACCAAGGGTC

GATACTCCCTGGACGTCTGGTCCTAA

Ec_POR (SEQ NO: 12):

ATGGGCGACTCCAACATGGACGCCTCCGCCCCCACCTCCGAGACTGTCGCTGAGGA

GGTCAGCTTATTTTCCATGATGGACATGTTCCTGTTCTCCCTGATCGTCGGCCTGCTG

ACCTACTGGTTCCTCTTCCGAAAGAAGAAGGACGAGATCCCCGAGTTCACCAAGAT

CCAGACCACCACCACCTCCGTCAAGGACTCCTCCTTCGTCGAGAAGATGAAGAAGA

CCGGCCGAAACATCATCGTCTTCTACGGCTCCCAGACCGGAACCGCCGAGGAGTTC

GCCAACCGACTCTCCAAGGACGCCCACCGATACGGCATGCGAGGTATGGCCGCCGA

CCCCGAGGAGTACGACCTCGCTGACCTCGGCTCCCTCTCCGAGATCGAGAACTCCCT

GGCCGTCTTCTGCATGGCCACCTACGGAGAGGGTGACCCCACCGACAACGCCCAGG

ACTTCTACGACTGGCTGCAGGAGGCCGACGTCGATCTGTCCGGCGTCAAGTACGCCG

TCTTCGGTCTGGGCAACAAGACCTACGAGCACTTTAACGCCATGGGCAAGTACGTCG

ATAAGCGACTGGAGCAGCTCGGTGCCCAGCGAATCTTCGAGCTGGGTCTGGGCGAC

GACGACGGTAACCTGGAGGAGGACTTCATCACCTGGCGAGAGCAGTTCTGGCCCGC

CGTGTGCGAGCACTTTGGAGTCGAGGCTACCGGCGAGGAGTCCTCCATTCGACAGT

ACGAGCTGCTGGTGCACACCGACATTGACGCCGCCAAGGTCTACGTGGGCGAGATG

GGCCGACTGAAGTCCTACGAGACTCAGAAGCCCCCCTTTGACGCCAAGAACCCCTTC

CTGGCCGTTGTCACCACCAACCGAAAGCTGAACCAGGGTACTGAGCGACACCTGAT

GCACCTGGAGCTGGACATCTCCGACTCCAAGATCCGATACGAGTCCCGGCGACCACG

TCGCCGTCTACCCCGCTAACGACTCCGCCCTGGTCAACCAGCTGGGCGAGATCCTCG

GCGCCGACCTCGACGTCATCATGTCCCTGAACAACCTCGACGAGGAGTCCAACAAG

AAGCACCCCTTCCCCTGCCCCACCTCCTACCGAACCGCCCTGACCTACTACCTGGAC

ATCACCAACCCCCCCCGAACCAACGTCCTCTACGAGCTGGCCCAGTACGCCTCCGAG

CCCTTCGAGCAGGAGCAGCTGCGAAAGATGGCCTCCTCCTCCGGCGAGGGCAAGGA

GCTGTACCTCACCTGGGTCGTCGAGGCCCGACGACACATTCTCGCCATCCTGCAGGA

CTACCCCTCCCTGCGACCCCCCATCGACCACCTGTGCGAGCTCCTGCCCGACTGCA

GGCCCGATACTACTCCATCGCCTCCTCCTCTAAGGTCCACCCCAACTCCGTCCACAT

CTGCGCCGTCGCCGTCGAGTACGAGACTAAGACCGGCCGAATTAACAAGGGCGTTG

CCACCACCTGGCTGCGAGCCAAGGAGCCCGCCAAGGAGAACGGCCGACGAGCCCTC

GTCCCCATGTTCGTGCGAAAGTCCCAGTTTCGACTGCCCTTCAAGGCCACCACCCCC

GTCATCATGGTCGGCCCCGGCACCGGAATCGCCCCTTTCATTGGCTTCATCCAGGAG

CGAGCCTGGCTGCAGCAGCAGGGCAAGGAGGTTGGCGAGACTCTCCTGTACTACGG

CTGCCGACGATCCGACGAGGACTACCTGTACCGAGATGAACTGGCCCAGTTCCACC

GAGATGGTTCTCTGACCCAGCTCAACGTGGCCTTCTCTCGAGAGCAGGCCCACAAGG

TCTACGTCCAGCACCTGCTGAAGCGAGACAAGGAGCACCTGTGGAAGCTGATCCAC

GAGGGCGGCGCCCACATTTACGTCTGCGGCGACGCCCGAAACATGGCCCGAGATGT

TCAGAACACCTTCTACGACATCGTCGCCGAGCTGGGAACCATGGAGCACGCCCAGG

CCGTCGATTACATTAAGAAGCTGATGACCAAGGGTCGATACTCCCTGGACGTCTGGT

CCTAA

Xl_POR (SEQ NO: 13):

ATGGGCGAGTCCTGCACCGAGCAGGACATGTGCACCTCCGAGCAGGGCAACGGCTC

CCCCGAGGAGGCTTTCTTCTCCATGGCCGACATGTTCCTGCTGTCCCTGATCGTCGGC

CTGCTGACCTACTGGTTTTTCTTTCGAAAGAAGAAGGAGGAGACTATCGAGTTCACC

AAGATCCAGCCCACCGTGAACAACTCCGTTCGAGAGTCCTCCTTTATCGAGAAGATG

AAGAAGACCGGCAAGAACATCGTCGTCTTCTACGGCTCCCAGACCGGCACCGGCGA

GGAGTTCGCCAACCGACTGGCCAAGGACGCCCACCGATACGGCGTCCGAGGAATGG

CCGCCGACCCCGAGGAGTTTCGAGATGGCCGACCTGTCCCGACTGACCGAGATTGAG

AACGCCCTGGCTGTCTCTGCATGGCCACCTACGGCGAGGGCGAACCCACCGACAA

CGCCCAGGACTTTTACGACTGGCTGCAGGAGACTGACATCGACCTGACCGGCCTGA

AGTACGCCGTTTTCGGACTGGGCAACAAGACCTACGAGCACTTTAACGCCATGGGC

AAGTACGTCGATAAGCGACTGGAGGAGCTGGGCGCCGAGCGAATCTTTGAGCTGGG

TATGGGCGACGACGACGGCAACCTGGAGGAGGACTTCATCACCTGGCGAGAGCAGT

TCTGGCCCGCCGTGTCGAGCACTTTGGTGTGGAGGCTACCGGAGAGGACTCCTCCA

TTCGACAGTACGAGCTGGTGGTGCACACCGACGAGAACATGAACAAGGTCTACACC

GGAGAGATGGGCCGACTGAAGTCCTACGAGACTCAGAAGCCCCCCTTCGACGCCAA

GAACCCCTTCCTGGCCAACGTCACCGTCAACCGAAAGCTGAACGAGGGCGGCGACC

GACACCTGATGCACCTGGAGCTGGACGTTACCGGCTCTAAGATCCGATACGAGTCC

GGAGATCATGTCGCCGTCTACCCCGCCAACGACACCGCCCTGGTCAACAAGCTGGG

AGAGATTCTGGGCGCCGACCTGGACACCGTGATTTCCCTGAACAACCTGGACGAGG

AGTCCAACAAGAAGCACCCCTTCCCCTGCCCCACCACCTACCGAACCGCCCTGACCT

ACTACCTGGACATCACCAACCCCCCCCGAACCAACGTCCTTCTACGAGCTGGCCCAGT

ACGCCACCGACTCCAAGGAGCAGGAGAACCTGCGAAAGATGGCCTCCTCCGCCCAG

GACGGCAAGGGCCTGTACCTCTCCTGGGTCGTCGAGTCCCGACGAAACATTCTGGCC

ATCCTGGAGGACGTGCCCTCCCTGCGACCCCCTCTGGACCACCTGTGCGAGCTGCTG

CCCCGACTGCAGGCTCGATACTACTCTATCGCCTCCTCCTCCAAGGTCCACCCTCCT

CCATCCACGTGTGCGCCGTCCTCGTCGAGTACGAGACTAAGACCGGCCGAGAGAAC

AAGGGCGTTGCCACCAACTGGCTCAAGAACAAGCAGCCCTCCGACAACGGCCACAA

GTCCTCCGTCCCCCATGTACGTTCGAAAGTCTCAGTTCCGACTCCCCTTCAAGCCCTCC

ACCCCCGTCATCATGATCGGCCCCGGCACCGGCATCGCCCCTTTCATCGGCTTTATC

CAGGAGCGAGAGTGGCTCAAGCAGCAGGGCAAGGACGTTGGCGAGACTGTCCTGTA

CTACGGCTGCCGACACGAGCACGAGGACTTTCTGTACAAGGACGAGCTGAAGCGAT

ACCACAAGGACGGCGTCCTGACCCAGCTCAACGTCGCCTTCTCCCGAGATCAAGAC

CGAAAGGTGTACGTCCAGCACCTGCTCAAGGACAACAAGGAGATGGTGTGGAAGCT

GATTCACGAGGACAACGCTCACATTTACGTCTGCGGCGACGCCCGAAACATGGCTC

GAGATGTTCAGAACACCTTCTACGACATCGTGGCCGAGTACGGCAAGATCGACCAC

GCCCAGGCCGTCGATTACATCAAGAAGCTGATGACCAAGGGACGATACTCCCAGGA

CGTTTGGTCCTAA

Oa_CYB5 (SEQ NO: 14):

ATGGCCGAGGAGTCTAGTAAACCAGTCAAGTACTACACCCTAGAGGAAATCCAGAA

GCACAACCACAGCAAATCGACCTGGCTGATTCTGCACTACAAGGTCTACGACCTCAC

AAAGTTCCTGGAAGAGCACCCGGGAGGAGAGGAGGTGCTTAGAGAGCAGGCTGGT

GGTGATGCAACTGAGAACTTTGAGGACGTTGGCCATTCAACGGATGCCCGAGAACT

TAGCAAGACCTTCATCATTGGCGAGCTGCATCCCGACGACCGGTCCAAGATCACCA

AGCCCTCTGAGTCCATCATCACAACTATTGACTCCAACTCGTCGTGGTGGACCAACT

GGCTCATTCCTGCCATTTCTGCTCTGGTGGTTGCGCTCATGTACCATTTGTATACTTC

TGAAAATTAA

Ma_CYB5 (SEQ NO: 15):

ATGGCCCGGCCAGGCAGACAAGGATGTCAAATACTATACGTTGGAAGAAATCCAGAA

GCACAAAGACTCCAAGTCTACGTGGGTCATTCTTCACCACAAGGTCTACGACCTGAC

CAAGTTTCTGGAGGAACATCCCGGTGGCGAGGAGGTACTTCGGGAGCAGGCTGGAG

GAGATGCCACCGAGAACTTTGAGGATTGTGGGCCACTCGACCGACGCTCGAGAGCTC

TCAAAGACATTCATCATTGGAGAGCTGCATCCTGACGACCGCAGCAAGATTGCCAA

GCCCAGCGAGAGTCTCATCACCACTGTGGAGTCCAACTCCTCTTGGTGGACCAACTG

GGTTATTCCGGCAGTTTCTGCGCTGGCCGTGGCTCTGATGTACCGACTCTACATGGG

CAGACGACTGACCTGTTTCTCGAAACCTGGAACAGGGGAGGGTCTGCCCCAACGAC

GAGGTGAAAAGAAGCCTGTGTTGATCACTTCGGCCGATAGAAATCTACCACTTAAG

GGCAAGTAA

Ec_CYB5 (SEQ NO: 16):

ATGGCCGAGCAGAGCGACAAGGCAGTCAAGTACTACACCCTCGAAGAGATCAAGA

AGCACAACCACTCGAAATCTACCTGGCTGATTCTGCACCACAAGGTCTATGACCTCA

CCAAGTTCCTGGAGGATCATCCAGGAGGAGAGGAGGTGCTTCGAGAACAGGCTGGT

GGTGATGCCACAGAGAACTTTGAGGATATTGGCCATTCTACAGACGCGAGAGAACT

TAGTAAAACGTTCATCATCGGCGAGCTGCATCCCGACGACCGGTCCAAGATTGCCA

AGCCCGTGGAGACTTTGATCACCACTGTGGACTCCAATTCATCGTGGTGGACCAACT

GGGTCATTCCTGCCATTTCTGCTGTAGTTGTTGCTCTCATGTACCGAATCTACACTGC

AGAAGATTAA

Ss_CYB (SEQ NO: 17):

ATGGCCGAGCAGTCCGACAAGGCCGTCAAGTACTACACCCTGGAGGAGATCCAGAA

GCACAACAACTCCAAGTCCACCTGGGCTGATCCTGCACCACAAGGTCTACGACCTGAC

CAAGTTCCTGGAGGAGCACCCCGGCGGTGAGGAGGTCCTGCGAGAGCAGGCCGGCG

GTGACGCTACCGAGAACTTCGAGGACGTCGGCCACTCCACCGACGCCCGAGAGCTG

TCCAAGACCTTTATCATTGGCGAGCTCCACCCCGACGACCGATCCAAGATCGCCAAG

CCCTCCGAGACTCTGATCACCACCGTCGAGTCCAACTCCTCCTGGTGGACCAACTGG

GTCATCCCCGCCATCTCCGCCCTGGTTGTCTCCCTGATGTACCACTTCTACACCTCCG

AGAACTAA

Hs_3β-HSD2(L236S)(SEQ NO: 18):

ATGGGTTGGTCCTGTCTGGTGACCGGAGCTGGTGGACTGCTGGGTCAGCGAATCGTG

CGACTGCTGGTCGAGGAGAAGGAGCTGAAGGAGATTCGAGCCCTGGACAAGGCTTT

CCGACCCGAGCTGCGAGAGGAGTTCTCTAAGCTGCAGAACCGAACCAAGCTGACCG

TGCTGGAGGGAGACATCCTGGACGAGCCCTTCCTGAAGCGAGCCTGTCAGGACGTC

TCCGTGGTCATTCACACCGATTGCATCATTGACGTGTTCGGCGTCACCCACCGAGAG

TCTATCATGAACGTGAACGTCAAGGGAACCCAGCTGCTGCTGGAGGCCTGTGTGCA

GGCTTCCGTGCCCGTCTTCATCTACACCTCTTCCATTGAGGTCGCCGGACCCAACTCT

TACAAGGAGATCATTCAGAACGGTCACGAGGAGGAGCCTCTGGAGAACACCTGGCC

TACCCCCTACCCCTACTCCAAGAAGCTGGCCGAGAAGGCTGTCCTGGCCGCTAACGG

CTGGAACCTGAAGAACGGAGACACCCTGTACACCTGCGCTCTGCGACCCACCTACA

TCTACGGAGAGGGTGGCCCCTTCCTGTCTGCCTCCATCAACGAGGCTCTGAACAACA

ACGGTATTCTGTCTTCCGTGGGCAAGTTCCTACCGTCAACCCCGTGTACGTCGGAA

ACGTGGCTTGGGCTCACATCCTGGCTTCGCGAGCTCTGCGAGATCCCAAGAAGGCCC

CCTCCGTCCGAGGACAGTTCTACTACATCTCCGACGACACCCCCCACCAGTCTTACG

ACAACCTGAACTACATTCTGTCTAAGGAGTTCGGTCTGCGACTGGACTCTCGATGGT

CCCTGCCCCTGACCCTGATGTACTGGATCGGCTTCCTGCTGGAGGTGGTGTCTTTCCT

GCTGTCCCCCATCTACTCTTACCAGCCCCCCTTCAACCGACACACCGTGACCCTGTCT

AACTCCGTCTTCACCTTCTCCTACAAGAAGGCCCAGCGGGACCTGGCTTACAAGCCC

CTGTACTCTTGGGAGGAGGCTAAGCAGAAGACCGTGGAGTGGGTCGGATCCCTGGT

GGACCGACACAAGGAGACTCTGAAGTCTAAGACCCAGTAA

Hs_3β-HSD2 (SEQ NO: 19):

ATGGGTTGGTCCTGTCTGGTGACCGGAGCTGGTGGACTGCTGGGTCAGCGAATCGTG

CGACTGCTGGTCGAGGAGAAGGAGCTGAAGGAGATTCGAGCCCTGGACAAGGCTTT

CCGACCCGAGCTGCGAGAGGAGTTCTCTAAGCTGCAGAACCGAACCAAGCTGACCG

TGCTGGAGGGAGACATCCTGGACGAGCCCTTCCTGAAGCGAGCCTGTCAGGACGTC

TCCGTGGTCATTCACACCGCTTGCATCATTGACGTGTTCGGCGTCACCCACCGAGAG

TCTATCATGAACGTGAACGTCAAGGGAACCCAGCTGCTGCTGGAGGCCTGTGTGCA

GGCTTCCGTGCCCGTCTTCATCTACACCTCTTCCATTGAGGTCGCCGGACCCAACTCT

TACAAGGAGATCATTCAGAACGGTCACGAGGAGGAGCCTCTGGAGAACACCTGGCC

TACCCCCTACCCCTACTCCAAGAAGCTGGCCGAGAAGGCTGTCCTGGCCGCTAACGG

CTGGAACCTGAAGAACGGAGACACCCTGTACACCTGCGCTCTGCGACCCACCTACA

TCTACGGAGAGGGTGGCCCCTTCCTGTCTGCCTCCATCAACGAGGCTCTGAACAACA

ACGGTATTCTGTCTTCCGTGGGCAAGTTCCTACCGTCAACCCCGTGTACGTCGGAA

ACGTGGCTTGGGCTCACATCCTGGCTCTGCGAGCTCTGCGAGATCCCAAGAAGGCCC

CCTCCGTCCGAGGACAGTTCTACTACATCTCCGACGACACCCCCCACCAGTCTTACG

ACAACCTGAACTACATTCTGTCTAAGGAGTTCGGTCTGCGACTGGACTCTCGATGGT

CCCTGCCCCTGACCCTGATGTACTGGATCGGCTTCCTGCTGGAGGTGGTGTCTTTCCT

GCTGTCCCCCATCTACTCTTACCAGCCCCCCTTCAACCGACACACCGTGACCCTGTCT

AACTCCGTCTTCACCTTCTCCTACAAGAAGGCCCAGCGGGACCTGGCTTACAAGCCC

CTGTACTCTTGGGAGGAGGCTAAGCAGAAGACCGTGGAGTGGGTCGGATCCCTGGT

GGACCGACACAAGGAGACTCTGAAGTCTAAGACCCAGTAA

Hs_3β-HSD1 (SEQ NO: 20):

ATGACCGGCTGGTCCTGCCTGGTGACCGGTGCTGGTGGCTTCCTGGGCCAGCGAATC

ATCCGACTGCTGGTCAAGGAGAAGGAGCTGAAGGAGATCCGAGTGCTGGACAAGGC

CTTCGGCCCCGAGCTGCGAGAGGAGTTTTTCCAAGCTGCAGAACAAGACCAAGCTGA

CCGTCCTGGAGGGCGACATCCTGGACGAGCCCTTTCTGAAGCGAGCCTGCCAGGAC

GTCAGCGTTATTATCCACACCGCCTGTATTATCGACGTCTTCGGCGTCACCCACCGA

GAGTCCATATGAACGTCAACGTCAAGGGAACCCAGCTGCTGCTGGAGGCCTGCGT

CCAGGCTTCCGTCCCTGTCTTCATCTACACCTCCTCCATCGAGGTCGCTGGCCCCAAC

TCCTACAAGGAGATCATCCAGAACGGCCACGAGGAGGAGCCCCTGGAGAACACCTG

GCCCGCTCCTTACCCCCACTCCAAGAAGCTCGCCGAGAAGGCCGTGCTGGCCGCTAA

CGGTTGGAACCTGAAGAACGGCGGCACCCTCTACACCTGTGCCCTGCGACCCATGTA

CATCTACGGTGAGGGCTCCCGATTCCTGTCCGCCTCCATTAACGAGGCTCTCAACAA

CAACGGCATCCTGTCCTCCGTCGGCAAGTTCTCCACCGTCAACCCCGTCTACGTCGG

CAACGTCGCCTGGGCTCACATCCTCGCCCTCCGAGCTCTGCAGGACCCTAAGAAGGC

CCCCTCCATTCGAGGTCAGTTCTACTACATCTCCGACGACACCCCCCACCAGTCCTA

CGACAACCTCAACTACACCCTCTCCAAGGAGTTCGGCCTGCGACTCGACTCCCGATG

GTCCTCCCCCTGTCCCTGATGTACTGGATCGGCTTCCTGCTGGAGATCGTCAGCTTT

TTACTGCGACCCATCTACACCTACCGACCCCCCTTCAACCGACACATCGTGACCTC

TCTAACTCCGTCTTCACCTTCTCCTACAAGAAGGCTCAGCGAGACTTGGCCTACAAG

CCCCTGTACTCCTGGGGAGGAGGCCAAGCAGAAGACCGTCGAGTGGGTCGGCTCTCT

GGTCGATCGACACAAGGAGACTCTGAAGTCCAAGACCCAGTAA

Hs_3β-HSD1(L237S)(SEQ NO: 21):

ATGACCGGCTGGTCCTGCCTGGTGACCGGTGCTGGTGGCTTCCTGGGCCAGCGAATC

ATCCGACTGCTGGTCAAGGAGAAGGAGCTGAAGGAGATCCGAGTGCTGGACAAGGC

CTTCGGCCCCGAGCTGCGAGAGGAGTTTTTCCAAGCTGCAGAACAAGACCAAGCTGA

CCGTCCTGGAGGGCGACATCCTGGACGAGCCCTTTCTGAAGCGAGCCTGCCAGGAC

GTCAGCGTTATTATCCACACCGCCTGTATTATCGACGTCTTCGGCGTCACCCACCGA

GAGTCCATATGAACGTCAACGTCAAGGGAACCCAGCTGCTGCTGGAGGCCTGCGT

CCAGGCTTCCGTCCCTGTCTTCATCTACACCTCCTCCATCGAGGTCGCTGGCCCCAAC

TCCTACAAGGAGATCATCCAGAACGGCCACGAGGAGGAGCCCCTGGAGAACACCTG

GCCCGTCCCTTACCCCCACTCCAAGAAGCTCGCCGAGAAGGCCGTGCTGGCCGCTAA

CGGTTGGAACCTGAAGAACGGCGGCACCCTCTACACCTGTGCCCTGCGACCCATGTA

CATCTACGGTGAGGGCTCCCGATTCCTGTCCGCCTCCATTAACGAGGCTCTCAACAA

CAACGGCATCCTGTCCTCCGTCGGCAAGTTCTCCACCGTCAACCCCGTCTACGTCGG

CAACGTCGCCTGGGCTCACATCCTCGCCTCCCGAGCTCTGCAGGACCCTAAGAAGGC

CCCCTCCATTCGAGGTCAGTTCTACTACATCTCCGACGACACCCCCCACCAGTCCTA

CGACAACCTCAACTACACCCTCTCCAAGGAGTTCGGCCTGCGACTCGACTCCCGATG

GTCCTCCCCCTGTCCCTGATGTACTGGATCGGCTTCCTGCTGGAGATCGTCAGCTTT

TTACTGCGACCCATCTACACCTACCGACCCCCCTTCAACCGACACATCGTGACCTC

TCTAACTCCGTCTTCACCTTCTCCTACAAGAAGGCTCAGCGAGACTTGGCCTACAAG

CCCCTGTACTCCTGGGGAGGAGGCCAAGCAGAAGACCGTCGAGTGGGTCGGCTCTCT

GGTCGATCGACACAAGGAGACTCTGAAGTCCAAGACCCAGTAA

Mm_3β-HSD (SEQ NO: 22):

ATGCCCGGATGGTCCTGTCTGGTGACCGGAGCTGGCGGATTCCTGGGTCAGCGAATC

ATTCAGCTGCTGGTGCAGGAGGAGGACCTGGAGGAGATTCGAGTGCTGGACAAGGT

CTTCCGACCCGAGACTCGAAAGGAGTTCTTCAACCTGGAGACTTCCATTAAGGTGAC

CGTCCTGGAGGGAGACATCCTGGACACCCAGTACCTGCGACGAGCCTGCCAGGGTA

TTTCTGTGGTCATCCACACCGCCGCTATCATTGACGTGACCGGCGTCATTCCCCGAC

AGACCATCCTGGACGTGAACCTGAAGGGAACCCAGAACCTGCTGGAGGCCTGTATT

CAGGCTTCCGTCCCCGCCTTCATCTTCTCTTCCTCTGTGGACGTCGCTGGTCCCAACT

CTTACAAGGAGATCGTGCTGAACGGCCACGAGGAGGAGTGCCACGAGTCCACCTGG

TCTGACCCCTACCCCTACTCCAAGAAGATGGCCGAGAAGGCTGTCCTGGCCGCTAAC

GGATCTATGCTGAAGAACGGTGGTACCCTGCAGACCTGTGCTCTGCGACCCATGTGC

ATCTACGGAGAGCGATCCCCCCTGATCTCTAACATCATTATCATGGCTCTGAAGCAC

AAGGGCATTCTGCGATCCTTCGGAAAGTTCAACACCGCCAACCCCGTGTACGTCGGA

AACGTGGCTTGGGCTCACATCCTGGCTGCTCGAGGTCTGCGAGATCCCAAGAAGTCT

CCCAACATTCAGGGCGAGTTCTACTACATCTCCGACGACACCCCCCACCAGTCTTTC

GACGAATTTCCTACACCCTGTCTAAGGAGTGGGGATTCTGTCTGGACTCCTCTTGG

TCCCTGCCTGTGCCTCTGCTGTACTGGCTGGCCTTCCTGCTGGAGACTGTGTCTTTCC

TGCTGTCTCCCATCTACCGATACATCCCCCCCTTCAACCGACACCTGGTGACCCTGTC

CGGTTCTACCTTCACCTTCTCCTACAAGAAGGCTCAGCGGGACCTGGGTTACGAGCC

TCTGGTGTCCTGGGAGGAGGCCAAGCAGAAGACCTCTGAGTGGATCGGCACCCTGG

TCGAGCAGCACCGAGAGACTCTGGACACCAAGTCTCAGTAA

At_3β-HSD (SEQ NO: 23):

ATGGCCGCTCCCGACTCTTCCATCAACAACCACCAGCTGCAGTACTCTGTGAACGTC

CAGGGAACCCAGAACGTCATCGACGCTTGTGTGGACGTCGGTGTGAAGCGACTGAT

CTACACCTCTTCCCCTTCTGTGGTCTTCGACGGCGTGCACGGAATCCTGAACGGCAC

CGAGTCCATGGCTTACCCCATTAAGCACAACGACTCTTACTCCGCTACCAAGGCCGA

GGGAGAGGAGCTGATTATGAAGGCCAACGGTCGAAACGGCCTGCTGACCTGTTGCA

TCCGACCCTCTTCCATTTTCGGTCCTGGCGACCGACTGCTGGTCCCTTCTCTGGTGGC

CGCTGCCCGAGCTGGCAAGTCCAAGTTCATCATTGGAGACGGTAACAACCTGTACG

ACTTCACCTACGTCGAGAACGTGGCTCACGCTCACGTCTGCGCTGAGCGAGCTCTGG

CTTCTGGAGGAGACGTGTCCACCAAGGCTGCCGGACAGGTGTTCGCCTTCTCCTAABt_3β-HSD (SEQNO: 24):

ATGGCCGGATGGTCTTGTCTGGTGACCGGTGGAGGTGGCTTCCTGGGTCAGCGAATC

ATTTGCCTGCTGGTCGAGGAGAAGGACCTGCAGGAGATCCGAGTGCTGGACAAGGT

CTTCCGACCCGAGGTGCGAGAGGAGTCTCTAAGCTGCAGTCCAAGATCAAGCTGA

CCCTGCTGGAGGGCGACATTCTGGACGAGCAGTGTCTGAAGGGAGCTTGCCAGGGT

ACCTCTGTGGTCATCCACACCGCCTCCGTGATTGACGTCCGAAACGCTGTCCCCGA

GAGACTATTATGAACGTGAACGTCAAGGGAACCCAGCTGCTGCTGGAGGCCTGTGT

GCAGGCTTCTGTGCCCGTCTTCATCCACACCTCCACCATTGAGGTCGCCGGTCCCAA

CTCTTACCGAGAGATCATTCAGGACGGCCGAGAGGAGGAGCACCACGAGTCTGCTT

GGTCTTCCCCCTACCCCTACTCCAAGAAGCTGGCCGAGAAGGCTGTGCTGGGTGCCA

ACGGCTGGGCTCTGAAGAACGGAGGTACCCTGTACACCTGTGCCCTGCGACCCATGT

ACATCTACGGCGAGGGATCTCCCTTCCTGTCCGCCTACATGCACGGCGCTCTGAACA

ACAACGGAATTCTGACCAACCACTGCAAGTTTCCCGAGTGAACCCCGTGTACGTCG

GTAACGTCGCTTGGGCTCACATCCTGGCTCTGCGAGCTCTGCGAGATCCCAAGAAGG

TGCCCAACATCCAGGGACAGTTCTACTACATTTCTGACGACACCCCCCACCAGTCCT

ACGACGACCTGAACTACACCCTGTCTAAGGAGTGGGGCTTCTGTCTGGACTCTCGAA

TGTCCCTGCCCATTTCCCTGCAGTACTGGCTGGCCTTCCTGCTGGAGATCGTCTCTTT

CCTGCTGTCCCCCATCTACAAGTACAACCCCTGCTTCAACCGACACCTGGTGACCCT

GTCTAACTCCGTCTTCACCTTCTCTTACAAGAAGGCTCAGCGGGACCTGGGTTACGA

GCCTCTGTACACCTGGGAGGAGGCTAAGCAGAAGACCAAGGAGTGGATCGGATCCC

TGGTGAAGCAGCACAAGGAGACTCTGAAGACCAAGATTCACTAA

Mt_3β-HSD (SEQ NO: 25):

ATGCTGCGACGAATGGGAGACGCCTCTCTGACCACCGAGCTGGGTCGAGTGCTGGT

CACCGGTGGAGCTGGTTTCGTCGGAGCTAACCTGGTGACCACCCTGCTGGACCGAG

GACACTGGGTCCGATCTTTCGACCGAGCTCCTTCCCTGCTGCCTGCTCACCCTCAGCT

GGAGGTGCTGCAGGGCGACATCACCGACGCTGACGTCTGTGCCCGCTGCCGTGGACG

GAATCGACACCATTTTCCACACCGCTGCCATCATTGAGCTGATGGGTGGCGCCTCTG

TCACCGACGAGTACCGACAGCGATCCTTCGCTGTGAACGTCGGAGGTACCGAGAAC

CTGCTGCACGCTGGACAGCGAGCTGGTGTCCAGCGATTCGTGTACACCTCTTCCAAC

TCCGTGGTCATGGGCGGACAGAACATTGCCGGTGGCGACGAGACTCTGCCCTACAC

CGACCGATTCAACGACCTGTACACCGAGACTAAGGGTGGTCGCCGAGCGATTCGTCCT

GGCTCAGAACGGTGTGGACGGCATGCTGACCTGCGCCATCCGACCCTCTGGAATTTG

GGGAAACGGTGACCAGACCATGTTCCGAAAGCTGTTCGAGTCCGTGCTGAAGGGTC

ACGTGAAGGTCCTGGTGGGCCGAAAGTCTGCTCGACTGGACAACTCCTACGTCCAC

AACCTGATCCACGGTTTCATTCTGGCTGCCGCTCACCTGGTGCCTGACGGTACCGCT

CCTGGACAGGCTTACTTCATCAACGACGCCGAGCCCATTAACATGTTCGAGTTCGCC

CGACCCGTCCTGGAGGCTTGTGGTCAGCGATGGCCCAAGATGCGAATCTCTGGCCCC

GCTGTCCGATGGGTCATGACCGGATGGCAGCGACTGCACTTCCGATTCGGTTTCCCT

GCTCCTCTGCTGGAGCCTCTGGCTGTGGAGCGACTGTACCTGGACAACTACTTCTCT

ATTGCCAAGGCTCGACGGGACCTGGGTTACGAGCCTCTGTTCACCACCCAGCAGGCT

CTGACCGAGTGCCTGCCCTACTACGTCTCCCTGTTCGAGCAGATGAAGAACGAGGCC

CGAGCTGAGAAGACCGCCGCTACCGTGAAGCCCTAA

Vv_3β-HSD (SEQ NO: 26):

ATGGCTGTGTACGCTGTCACCGGTGGAGCTGGATTCCTGGGTCGATACATCGTGAAG

CTGCTGATTTCCGCTGACGACGTCCAGGAGATCCGAGTGATCGACATTGTCGAGGAC

CCCCAGCCCATTACCTCTAAGGTGAAGGTCATCAACTACATTCAGTGTGACATCAAC

GACTTCGACAAGGTGCGAGAGGCCCTGGACGGTGTCAACCTGATCATTCACACCGC

CGCTCTGGTGGACGTCTTCGGCAAGTACACCGACAACGAGATCATGAAGGTGAACT

ACTACGGAACCCAGACCATTCTGGCCGCTTGCGTCGACCTGGGTATCAAGTACCTGA

TCTACACCTCTTCCATGGAGGCCATTGGTCCCAACAAGCACGGCGACCCCTTCATCG

GACACGAGCACACCCTGTACGACATTTCCCCCGGACACGTGTACGCCAAGTCTAAG

CGAATGGCTGAGCAGCTGGTCATGAAGGCCAACAACTCCGTCATCATGAACGGCGC

TAAGCTGTACACCTGTTGCCTGCGACCCACCGGAATCTACGGAGAGGGCGACAAGC

TGACCAAGGTCTTCTACGAGCAGTGTAAGCAGCACGGAAACATCATGTACCGAACC

GTGGACGACGACCGCTGTCCACTCCCGAGTGTACGTCGGTAACGTGGCTTGGATGCAC

GTCCTGGCCGCTAAGTACATCCAGTACCCCGGTTCTGAGATTAAGGGCAACGCCTAC

TTCTGTTACGACTACTCTCCCTCCTGCTCTTACGACATGTTCAACCTGCTGCTGATGA

AGCCCCTGGGCATCGAGCAGGGATCTCGAATTCCCCGATGGATGCTGAAGATGTAC

GCTTGCAAGAACGACATGAAGCGAATCCTGTTCCGAAAGCCCTCCCTGCTGAACAA

CTACACCCTGAAGATTTCTAACACCACCTTCGAGGTGCGAACCAACAACGCCGAGCT

GGACTTCAACTACTCCCCATTTTCAACGTGGACGTCGCTTTCGAGCGAACCCGAAA

GTGGCTGGAGGAGTCTGAGTAA

3β-HSD of Homo sapiens (type I, L237S mutation), the mutation site is underlined AA (SEQ NO: 27):

3β-HSD of Homo sapiens (type II, L236S mutation), the mutation site is underlined AA (SEQ NO: 28):

The raw materials and reagents used in the directional synthesis of the network path product provided by the present invention can all be purchased from the market.

The present invention will be further described below in conjunction with embodiments:

Example 1: Heterologous characterization of substrate specificity of key components of the 4AD pathway (3β-HSD) and directed synthesis of progesterone

1. Obtaining chassis strains

The wild-type Yarrowia lipolytica strain number is ATCC201249 and was provided by Yuan Yingjin's research group. It was mentioned in the paper Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

2. Acquisition of exogenous functional gene elements

The sources of the genes CYP17A1 (17-alpha-hydroxylase/17, 20-lyase), POR (NADPH-cytochrome P450 reductase), 3β-HSD (3β-hydroxysteroid dehydrogenase), CYB5 (cytochrome B5, Cytochrome b5), and mCYP11A1 (mature P450scc) involved in the present invention are shown in Table 3.

Table 3 Sources of genes involved in the present invention

The above four component genes used in the present invention are all obtained by artificial synthesis by adding 5' gcggccgcggtctcca (as shown in SEQ NO: 1) and 3' taaaggagaccgcggccgc (as shown in SEQ NO: 2) to both ends of the gene after codon optimization of Yarrowia lipolytica and appropriate avoidance of common restriction enzyme sites. The synthesis pathway of androstenedione from pregnenolone is shown in Figure 1, and the synthesis pathway includes the steps of synthesizing progesterone from pregnenolone (Figure 2), synthesizing dehydroepiandrosterone from pregnenolone (Figure 3), synthesizing 17-hydroxyprogesterone from pregnenolone (Figure 4), and synthesizing 17-hydroxypregnenolone from pregnenolone (Figure 5).

3. Test methods:

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder;

Biotransformation medium: 20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract powder.

Steroid substrate stock solution: 1.75 g/L (50% EtOH-Tween80) P4 solution, 1.75 g/L (50%

EtOH-Tween80)17OHP4 solution

3β-HSD isomerization transformation experiment: The recombinant strains RS3B-1 to RS3B-9 (see the construction of modular integration plasmids below) were inoculated into 5 mL of seed culture medium, cultured at 30°C and 220 rpm for 14 to 16 h, and inoculated into 5 mL of biotransformation medium with an initial cell concentration of _OD600 = 0.2, cultured at 28°C and 220 rpm for 24 h, and 150 μL of P4 solution and 17OHP4 solution were added respectively and incubated for another 40 h to obtain fermentation broth, and the steroid products were quantified by the following method.

Pregnenolone quantitative method: Take 1mL of fermentation broth, centrifuge at 12000g for 2min to collect the bacteria, and wash twice with water. Add 1mL of 3mol/L hydrochloric acid to resuspend the bacteria, boil in 100℃ boiling water for 5min, and centrifuge at 12000rpm for 1min to collect the cell precipitate. Wash the cell precipitate 3 times with 1mL of distilled water. Add 420μL of 2mol/L potassium hydroxide-methanol solution to the broken cell precipitate and react in a 37℃ constant temperature incubator for 2h. Take out the saponification reaction centrifuge tube, cool to room temperature (25℃±5℃), add an equal volume of n-hexane, vortex for 10min, centrifuge at 12000rpm for 1min, and take the upper n-hexane phase to a new centrifuge tube. Repeat the extraction of the lower layer with n-hexane once, and combine the two n-hexane phases. The n-hexane phase was concentrated using a vacuum centrifugal concentrator (when the sample solvent was n-hexane: 25°C, 30 min, 7000 rpm). The solid remaining in the tube after concentration was steroidal substances. 100 μL MSTFA was added and reacted at 37°C for 2 h. 100 μL n-hexane was added and the mixture was filtered and detected by gas mass spectrometry.

Quantitative method for progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, DHEA, and androstenedione: Take 1 mL of fermentation broth, add glass beads and 700 μL ethyl acetate, shake and extract for 10 minutes, collect the upper organic phase and re-extract the aqueous phase with fresh ethyl acetate, combine the two extracted organic phases, and concentrate the liquid with a vacuum centrifugal concentrator (when the sample solvent is ethyl acetate: 25°C, 1200min, 7000rpm). The solid remaining in the tube after concentration is steroidal substances. Add 100 μL MSTFA and react at 37°C for 2 hours, add 100 μL n-hexane and filter. P5, P4, DHEA, 4AD, 17OHP4, and 17OHP5 are detected under GC-MS. (The yield of 17OHP4 and 17OHP5 is relatively quantified using the P5 standard curve)

4. Construction of modular integration plasmids

Construction of module three: There are two types of 3β-HSD with different catalytic specificity in the human body: Type I 3β-HSD has higher activity and uses DHEA as the main substrate, and Type II 3β-HSD tends to use P5 and 17OHP5 as substrates. In the present invention, the above two human 3β-HSDs are abbreviated as Hs_3β-HSD1 and Hs_3β-HSD2 respectively after codon optimization. In 1999, Moisan et al. pointed out that the L236S mutation of Hs_3β-HSD2 can increase the maximum reaction rate of the protein, and the mutant protein is abbreviated as Hs_3β-HSD2mut. After comparison with the original sequence, the present invention also introduced the corresponding mutation L237S into Hs_3β-HSD1 and named it Hs_3β-HSD1mut. This example also selected 3β-HSD from five different species-representative sources, namely Mm_3β-HSD from mouse Mus musculus, 3β-HSD2 from cattle Bos taurus, 3β-HSD from Vaccinia virus, 3β-HSD from Mycobacterium tuberculosis, and At_3β-HSD from Arabidopsis thaliana. After codon optimization, they are abbreviated as Mm_3β-HSD, Bt_3β-HSD, Vv_3β-HSD, Mt_3β-HSD and At_3β-HSD, respectively. In order to facilitate integration into the vector pINA1269-Nat by Gibson assembly (the LEU2 marker between the BglII and ClaI restriction sites of the commercial vector plasmid pINA1269 was replaced with the vector plasmid after the nourseoinhibin resistance marker Nat was replaced), a 21 bp homology arm sequence gggaacccgaaactaaggatc (as shown in SEQ NO: 3) upstream of the vector BamHI site was introduced at the 5' end of the above gene by PCR reaction, and a 21 bp homology arm sequence gtacctccatggcctgtcccc (as shown in SEQ NO: 4) downstream of the vector KpnI site was introduced at the 3' end of the gene. After assembly with the linearized vector of BamHI and KpnI, 9 corresponding pINA1269-Nat-3β-HSD integration recombinant plasmids pRS3B-1 to pRS3B-9 (i.e., pINA1269-Nat-Mm_3β-HSD, pINA1269-Nat-Bt_3β-HSD, pINA1269-Nat-Vv_3β-HSD, pINA1269-Nat-At_3β-HSD, pINA1269-Nat-Mt_3β-HSD, pINA1269-Nat-Hs_3β-HSD1, pINA1269-Nat-Hs_3β-HSD2, pINA1269-Nat-Hs_3β-HSD1mut, and pINA1269-Nat-Hs_3β-HSD2mut), i.e., module three, were obtained. The plasmids constructed above were transformed into competent E. coli DH5α, colony PCR screening was performed, and the plasmids were extracted for single and double restriction enzyme verification and sequencing verification to ensure that the target fragments were correctly connected and the base sequence had not mutated. pINA1269 is the name of the integrative plasmid. After linearization by NotI, the plasmid can be used to integrate into the pBR322 site of the yeast genome, where pBR322 is the name of the integration site.

5. Experimental results

The conversion efficiency of steroidal substrates was characterized by the amount of corresponding isomers synthesized. The results of 3β-HSD whole cell catalysis are shown in FIG6 .

From the whole cell conversion results of the three substrates, we can see that: (1) In the conversion experiment with pregnenolone (P5) as the substrate, Vv_3β-HSD showed the strongest pregnenolone conversion efficiency, reaching 6.8%. Type I human and mutant 3β-HSD showed the second strongest catalytic efficiency, reaching 4.6-4.8% (2) For the conversion experiment with 17-hydroxypregnenolone (17OHP5) as the substrate, the four human 3β-HSDs and mutants all showed strong conversion efficiency, among which type I human 3β-HSD had the highest progesterone conversion rate of 3.1%. (3) When dehydroepiandrosterone (DHEA) was used as the substrate, type II human, bovine, and mycobacterium sources all showed strong catalytic activity, among which type II human 3β-HSD had a conversion efficiency of up to 10.5%.

By expressing heterologous 3β-HSD alone in a wild-type yeast chassis, the present invention achieves the synthesis of 4AD pathway node product: P4 using P5 as substrate. In addition, the whole-cell catalytic efficiency of 3β-HSD from different sources shows strong differences in substrate preference: (1) Several 3β-HSDs except human and low-catalytic Arabidopsis sources all show strong substrate preference for pregnenolone, while the conversion ability for 17OHP5 is relatively low; (2) Different from the substrate preference of 3β-HSD in human cells, human 3β-HSD in the Yarrowia lipolytica system generally shows a relatively balanced catalytic ability for three steroid substrates. In particular, wild-type II human 3β-HSD shows a higher DHEA conversion ability than type I human 3β-HSD.

Example 2: Heterologous Characterization of Substrate Specificity of Key Components of 4AD Pathway (CYP17A1) and Directed Synthesis of 17-Hydroxypregnenolone

1. Acquisition of experimental materials

CYP17 hydroxylase is a key node in the steroid anabolic pathway, catalyzing the 17α-hydroxylation and 17,20-cleavage reactions of C21 steroids as substrates. The participation of cytochrome b5 (CYB5) promotes the 17,20-lyase activity of CYP17. The catalytic activity of CYP17A1 has strong species specificity. According to the substrate catalytic specificity, CYP17A1 is usually divided into △ ^4,5 type, △ ⁵ type and △ ⁴ type. The selection of CYP17 in this study was based on the in vitro catalytic activity parameters of CYP17 from multiple sources compiled by Gilep and his colleagues. Since the catalytic activity of the only △ ⁴ type CYP17 reported was weak, based on the in vitro enzyme activity parameters and protein evolution relationship, the present invention only selected the △ ⁵ type CYP17A1 from sheep (Ovis aries) and the △ ^{4, 5} type CYP17A1 from golden hamsters (Mesocricetus auratus), horses (Equus caballus), and African clawed frogs (Xenopus laevis), and after codon optimization, they were abbreviated as Oa_CYP17A1, Ma_CYP17A1, Ec_CYP17A1, and Xl_CYP17A1, respectively. Similarly, the above-mentioned CYP17A1 original ligand NADPH-cytochrome P450 reductase (POR) was also abbreviated as Oa_POR, Ma_POR, Ec_POR, and Xl_POR after codon optimization. Considering that the 17,20-lyase activity of CYP17 is affected by CYB5, CYB5 from sheep, golden hamster, horse, and pig (Sus scrofa) sources were introduced into the test, and the four CYB5s were abbreviated as Oa_CYB5, Ma_CYB5, Ec_CYB5, and Ss_CYB5 after codon optimization.

The wild type Yarrowia lipolytica was obtained as described in Example 1.

Construction of module one: The left arm of the IntD integration site and the Saccharomyces cerevisiae GPM1t terminator were spliced together by the OE-PCR method; the 40bp terminal sequence of the Saccharomyces cerevisiae FBA1t terminator, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, and the right arm of the IntD integration site were spliced together by the OE-PCR method to obtain fragments containing NotI restriction sites at both ends, named IntD-L and IntD-R, respectively; then, the artificially synthesized CYP17A1 and POR from four different sources were connected with the expression modules TEF1inp-LIP2t-GPDt and GPDt-TEF1inp-OCT1t-FBA1t after digestion with BsmBI to obtain integration plasmids, and the CYP17A1 and POR modules with the same species source were assembled with pUC18H after digestion with IntD-L, IntD, and HincII by Gibson to obtain integration plasmids, and module one was obtained after NotI digestion. Construction of module 2: The left arm of the IntB integration site, the auxotrophic uracil tag Ura3, the right arm of the IntB integration site, the promoter TEF1in, the terminator ACOt and the CYB5 from four sources were spliced together by the Gibson method to obtain an integration plasmid, and module 2 was obtained after NotI digestion. The module integration plasmids constructed above were transformed into Escherichia coli competent DH5α, colony PCR screening, and plasmid extraction for single and double enzyme digestion verification and sequencing verification to ensure that the target fragment was correctly connected and the base sequence had not mutated.

Construction of module four: The left arm of the IntF integration site, pUC18H after HincII digestion, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, the right arm of the IntF integration site, CYP17A1 and POR from Equus caballus (the method is the same as that of module one) were spliced together by the Gibson method to obtain a fragment containing NotI digestion sites at both ends, and module four was obtained after NotI digestion.

The 17-hydroxylation transformation experiment verified the construction of the strain, and the modules containing four genes from different sources were respectively integrated into ATCC201249 to obtain strains SyBE_Yl2091001~SyBE_Yl2091004.

The construction of the strain was verified by 17,20-lysis transformation experiments, and module 2 containing 4 genes from different sources was respectively integrated into the above-constructed strains SyBE_Yl2091001~SyBE_Yl2091004 to obtain strains SyBE_Yl2091005~SyBE_Yl2091016 and SyBE_Yl2090013~SyBE_Yl2090016.

2. Experimental methods

Biotransformation medium: 20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract powder;

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder;

YPD fermentation medium: 50g/L glucose, 20g/L peptone, 10g/L yeast extract powder.

Steroid substrate stock solution: 1.75 g/L (50% EtOH-Tween80) P5 solution, 1.75 g/L (50%

EtOH-Tween80) P4 solution, 1.75g/L (50% EtOH-Tween80) 17OH P5 solution, 1.75g/L (50%

EtOH-Tween80)17OHP4 solution

17-Hydroxylation conversion experiment: SyBE_Yl2091001~SyBE_Yl2091004 were inoculated into 5 mL seed culture medium, cultured at 30°C, 220 rpm for 14-16 h, inoculated into 5 mL biotransformation medium with an initial bacterial concentration of _OD600 = 0.2, cultured at 28°C, 220 rpm for 24 h, 150 μL of P4 and P5 substrate mother solutions were added respectively and incubated for another 16 h, and 1 mL of sample was taken to detect the 17OHP4 content according to the method in Example 1.

17,20-cleavage conversion experiment: SyBE_Yl2091005~SyBE_Yl2091016 were inoculated into 5 mL of seed culture medium, cultured at 30°C and 220 rpm for 14-16 h, and inoculated into 5 mL of biotransformation medium with an initial bacterial concentration of _OD600 = 0.2, cultured at 28°C and 220 rpm for 24 h, and 150 μL of 17OHP4 and 17OHP5 substrate mother solutions were added respectively and incubated for another 144 h. 1 mL of sample was taken and the DHEA or 4AD content was detected according to the method in Example 1.

3. Experimental results

17-Hydroxylation conversion experiment: By expressing heterologous 3β-HSD alone in the wild-type yeast chassis, the present invention realizes the synthesis of 4AD pathway node product: 17OHP5 using P5 as substrate. It can be seen from the experimental results (Figure 7) that the 17α-hydroxylation activity of sheep, golden hamster, and African clawed frog CYP17A1 for progesterone is 1.2 to 14.5 times that of pregnenolone as substrate. Among the tested △ 4 ^{and 5} type CYP17A1, although the golden hamster CYP17A1 showed the strongest 17α-hydroxylation activity under the conditions of pregnenolone and progesterone as substrates, it was only 39.2% and 13.8% of the activity of △ ⁵ type sheep CYP17A1, respectively. CYP17A1 from horses and African clawed frogs showed very weak catalytic activity for both substrates.

17,20-cleavage conversion experiment: As shown in the experimental results (Figure 8), all tested strains showed 17,20-lyase activity for both substrates. (1) For the three tested △ ^4,5 type CYP17A1: the engineered bacteria containing African clawed frog CYP17A1 all showed low 17,20-lyase activity, indicating that the CYP17 from this source cannot effectively achieve functional expression under the current system; for golden hamster and horse CYP17A1, the participation of CYB5 can significantly improve the 17,20-lyase activity. (2) For △ ⁵ type sheep CYP17A1: the protein from this source has a stronger substrate preference and biotransformation ability for 17-hydroxypregnenolone than 17-hydroxyprogesterone; the 17,20-lyase activity of the protein from this source for 17α-hydroxyprogesterone has a strong CYB5 source dependence. (3) Regarding CYB5: In most cases, CYB5 can promote the 17,20-lyase activity of CYP17A1. As highly efficient ligands, CYB5 from pigs and horses, when adapted to CYP17A1 from multiple sources, can often promote the latter's conversion efficiency of 17α-hydroxyprogesterone to a greater extent.

Regarding the selection of enzyme sources for the reconstruction of the heterologous 4AD pathway of Yarrowia lipolytica, the present invention summarizes that: (1) for the isomerization (dehydrogenation) reaction, Vv_3β-HSD is the preferred protein for catalyzing the reaction with P5 as the substrate; (wild type) type I and type II Hs_3β-HSD are the preferred proteins for catalyzing the reaction with 7OHP5 and DHEA as the substrates; (2) when P5 and P4 are used as substrates, Oa_CYP17A1 is the most preferred source for catalyzing the 17α-hydroxylation reaction; (3) for the 17,20-cleavage reaction with 17α-hydroxypregnenolone as the substrate, although the sheep-derived protein has high catalytic activity, the △ ^4,5 -type golden hamster-derived CYP17A1 is also a reliable alternative source; for the 17,20-cleavage reaction with 17α-hydroxyprogesterone as the substrate, the horse-derived CYP17A1 can achieve efficient catalysis regulated by CYB5; at the same time, the pig, golden hamster and horse-derived CYB5 can all be used as efficient ligands for pathway construction.

Example 3: Synthesis of DHEA using P5 as substrate and synthesis of 4AD using P4 as substrate

1. Acquisition of experimental materials

The acquisition of strains SyBE_Yl2091005~SyBE_Yl2091016 is the same as described in Example 2.

The acquisition of module 4 is the same as described in Example 2.

Construction of module 5: The Leu2 screening marker of SyBE_Y12091004 was knocked out using the Cre-loxP system to obtain SyBE_Y12091004 without Leu2 tag. Module 4 linearized by NotI and module 2 containing horse-derived Ec_CYB5 were integrated into the genome of SyBE_Y12091004 without Leu2 tag through yeast transformation to obtain strain SyBE_Y12091030.

2. Experimental methods

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder;

Biotransformation medium: 20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract powder.

Steroid substrate stock solution: 3.5 g/L (50% EtOH-Tween80) P5 solution, 3.5 g/L (50% EtOH-Tween80) P4 solution

DHEA synthesis experiment: SyBE_Yl2091030 (experimental group) and SyBE_Yl2091016 (control) were inoculated into 5 mL of seed culture medium and cultured at 30°C and 220 rpm for 14 to 16 h. They were inoculated into 5 mL of biotransformation medium with an initial cell concentration of _OD600 = 0.2 and cultured at 28°C and 220 rpm for 24 h. 150 μL of P5 substrate mother solution was added and the culture was continued for 120 h. 1 mL of sample was taken and the DHEA content was detected according to the method in Example 1.

4AD synthesis experiment: SyBE_Yl2091030 (experimental group) and SyBE_Yl2091016 (control) were inoculated in 5 mL of seed culture medium, cultured at 30°C and 220 rpm for 14-16 h, inoculated in 5 mL of biotransformation medium with an initial bacterial concentration of _OD600 = 0.2, cultured at 28°C and 220 rpm for 24 h, 150 μL of P4 substrate mother solution were added and incubated for another 120 h, and 1 mL of sample was taken to detect the 4AD content according to the method in Example 1.

3. Experimental results

In this example, Oa_CYP17A1 (strong 17α-hydroxylation activity), Ec_CYP17A1 (strong 17,20-cleavage activity), and Ec_CYB5 (which has a strong promoting effect on the 17,20-cleavage activity of both Oa_CYP17A1 and Ec_CYP17A1) were combined and characterized in strain SyBE_Yl2091030 to achieve efficient conversion of P5/P4 to DHEA/4AD.

DHEA synthesis experiment: In the biotransformation with P5 as substrate, the DHEA synthesis amount of SyBE_Yl2091030 reached 12.6 mg/L, which was 7.32 times higher than that of the control group SyBE_Yl2091016 (Figure 9).

4AD synthesis experiment: In the biotransformation with P4 as the substrate, strain SyBE_Yl2091030 achieved an androstenedione synthesis of 13.9 mg/L, which was 86.2 times higher than that of the control strain SyBE_Yl2091016 (Figure 10).

In this embodiment, the present invention realizes the directed synthesis of DHEA using P5 as a substrate and the directed synthesis of 4AD using P4 as a substrate, and improves the catalytic efficiency by combining and expressing the high-efficiency pathway component proteins.

Example 4: Synthesis of 17OHP4 using P5 as substrate

The synthesis of 17OHP4 from P5 involves two types of reactions: 17α-hydroxylation reaction with P4 or P5 as substrate, and isomerization reaction with P5 or 17OHP5 as substrate. The present invention selects Ma_CYP17A1 to catalyze two 17α-hydroxylation reactions, and selects Hs_3β-HSD2, which has strong catalytic specificity for two intermediates (P5 and 17OHP5), and Vv_3β-HSD, which has strong catalytic specificity for P5. The above three highly efficient catalytic proteins are used together for the construction of the 17OHP4 pathway.

1. Acquisition of experimental materials

The construction of modules 1, 2, 3 and 4 is the same as that described in Examples 1 and 2.

Construction of module six: The left arm of the IntC integration site, the artificial promoter hp8d, Hs_3β-HSD2, the Yarrowia lipolytica terminator OCTt, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, and the right arm of the IntC integration site were spliced together by OE-PCR to obtain a fragment containing NotI restriction sites at both ends; the above fragment was connected with the plasmid pUC57-Kan-Simple linearized by HindIII to obtain an integration plasmid, namely module six. The module integration plasmids constructed above were transformed into Escherichia coli competent DH5α, colony PCR screening, and plasmid extraction for single and double restriction enzyme verification and sequencing verification to ensure that the target fragment was correctly connected and the base sequence had not mutated.

Construction of module seven: Module one (pIntD-Ma_CYP17-POR) expressing CYP17A1-POR from golden hamster (Mesocricetus auratus), module six, and module three expressing Vv_3β-HSD were simultaneously integrated into ATCC201249 to obtain the recombinant lipolytic yeast strain SyBE_Yl2090007.

2. Experimental methods

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder;

Biotransformation medium: 20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract powder.

Steroid substrate stock solution: 3.5 g/L (50% EtOH-Tween80) P5 solution.

4AD synthesis experiment: SyBE_Yl2090007 was inoculated into 5 mL seed culture medium, cultured at 30°C and 220 rpm for 14-16 h, inoculated into 5 mL biotransformation culture medium with an initial bacterial concentration of OD ₆₀₀ = 0.2, cultured at 28°C and 220 rpm for 24 h, added with 150 μL P5 substrate mother solution and continued to incubate for 120 h, and 1 mL sample was taken to detect the 17OHP4 content according to the method in Example 1. (17OHP4 was relatively quantified using the P5 standard curve)

3. Experimental results

The synthesis amount of 17OHP4 in strain module 7 reached 3.90 mg/L (Figure 11). In this embodiment, the present invention realizes the directional synthesis of 17OHP4 using P5 as substrate, and improves the catalytic efficiency by combining and expressing the high-efficiency pathway component proteins.

Example 5: De novo synthesis of progesterone using glucose

1. Obtaining chassis strains

Provided by Yuan Yingjin's research group, the high-yield campesterol Yarrowia lipolytica chassis strain number is SyBE_Yl2060077, and the wild-type Yarrowia lipolytica strain number is ATCC201249. SyBE_Yl2060077 strain is mentioned in the literature Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System.

2. Acquisition of exogenous functional gene elements

The sources of the genes CYP17A1 (17-alpha-hydroxylase/17, 20-lyase), POR (NADPH-cytochrome P450 reductase), 3β-HSD (3β-hydroxysteroid dehydrogenase), CYB5 (cytochrome B5, Cytochrome b5), and mCYP11A1 (mature P450scc) involved in the present invention are shown in Table 4.

Table 4 Sources of genes involved in the present invention

The above four component genes used in the present invention are all obtained by artificial synthesis after Yarrowia lipolytica codon optimization and appropriate avoidance of common restriction enzyme cutting sites, and additional 5' gcggccgcggtctcca (as shown in SEQ NO: 1) and 3' taaaggagaccgcggccgc (as shown in SEQ NO: 2) are added to both ends of the gene.

3. Test methods:

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder;

YPD fermentation medium: 50g/L glucose, 20g/L peptone, 10g/L yeast extract powder.

Quantitative method for pregnenolone (P5) and campsterol: Take 1 mL of fermentation broth, centrifuge at 12000g for 2 minutes to collect the cells, and wash twice with water. Add 1 mL of 3 mol/L hydrochloric acid to resuspend the cells, boil them in 100℃ boiling water for 5 minutes, and centrifuge at 12000rpm for 1 minute to collect the cell precipitate. Wash the cell precipitate 3 times with 1 mL of distilled water. Add 420 μL of 2 mol/L potassium hydroxide-methanol solution to the broken cell precipitate and react in a 37℃ constant temperature incubator for 2 hours. Take out the saponification reaction centrifuge tube, cool it to room temperature (25℃±5℃), add an equal volume of n-hexane, vortex and shake for 10 minutes, centrifuge at 12000rpm for 1 minute, and take the upper n-hexane phase to a new centrifuge tube. Repeat the extraction of the lower layer with n-hexane once, and combine the two n-hexane phases. The n-hexane phase was concentrated using a vacuum centrifugal concentrator (when the sample solvent was n-hexane: 25°C, 30 min, 7000 rpm). The solid remaining in the tube after concentration was steroidal substances. 100 μL MSTFA was added and reacted at 37°C for 2 h. 100 μL n-hexane was added and the mixture was filtered and detected by gas mass spectrometry.

Quantitative method for progesterone, 17-hydroxypregnenolone (17OHP5), 17-hydroxyprogesterone (17OHP4), dehydroepiandrosterone, androstenedione, and testosterone: Take 1 mL of fermentation broth, add glass beads and 700 μL ethyl acetate, shake and extract for 10 min, collect the upper organic phase and re-extract the aqueous phase with fresh ethyl acetate, combine the two extracted organic phases, and concentrate the liquid with a vacuum centrifugal concentrator (when the sample solvent is ethyl acetate: 25°C, 1200 min, 7000 rpm). The solid remaining in the tube after concentration is the steroidal substance. For progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (4AD), and testosterone (TS) samples, add 100 μL MSTFA and react at 37°C for 2 h, add 100 μL n-hexane and filter, and then detect by gas mass spectrometry. For 17-hydroxypregnenolone and 17-hydroxyprogesterone samples, dissolve in 200 μL anhydrous ethanol, filter, and then detect by ultra-high performance liquid chromatography.

4. Construction of modular integration plasmids

For the construction of upstream module strains, mCYP11A1 was expressed under the TEF1p promoter, 3β-HSD was expressed under the EXP1p promoter, and both were integrated into the pBR322 site. For the construction of downstream module strains, CYP17A1 and POR were expressed under the promoter TEF1inp and integrated into the IntD site of the chassis strain genome.

Construction of module A: The left arm of the IntD integration site and the Saccharomyces cerevisiae GPM1t terminator were spliced together by the OE-PCR method; the 40bp terminal sequence of the Saccharomyces cerevisiae FBA1t terminator, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, and the right arm of the IntD integration site were spliced together by the OE-PCR method to obtain fragments containing NotI restriction sites at both ends, named IntD-L and IntD-R, respectively; then, the artificially synthesized CYP17A1 and POR from four different sources were connected with the expression modules TEF1inp-LIP2t-GPDt and GPDt-TEF1inp-OCT1t-FBA1t after digestion with BsmBI to obtain integration plasmids, and the CYP17A1 and POR modules with the same species source were assembled with pUC18H digested with IntD-L, IntD, and HincII by Gibson to obtain integration plasmids, and module A was obtained after NotI digestion. Construction of module B: The left arm of the IntB integration site, the defective uracil tag Ura3, the right arm of the IntB integration site, the promoter TEF1in, the terminator ACOt and the CYB5 from three sources were spliced together by the Gibson method to obtain an integration plasmid, and module B was obtained after NotI digestion. Construction of module C: The porcine mCYP11A1 was integrated into the expression cassette with the TEF1p promoter, and the 3β-HSD from six different sources was integrated into the expression cassette with the EXP1p promoter, and the mCYP11A1 expression cassette and the six different 3β-HSD expression cassettes were assembled by Gibson into the pINA1269 integration plasmid after SalI and ClaI digestion, and finally the plasmid was linearized after NotI digestion to obtain module C. The module A to C integration plasmids constructed above were transformed into competent E. coli DH5α, respectively, and colony PCR was performed for screening. The plasmids were extracted for single and double restriction enzyme digestion verification and sequencing verification to ensure that the target fragments were correctly connected and the base sequence had not mutated.

5. Experimental results

Upstream modules: By integrating 6 different modules C into yeast SyBE_Yl2060077, upstream modules SyBE_Yl2090018, SyBE_Yl2090006, SyBE_Yl2091025 to SyBE_Yl2091028 were obtained

First, the present invention obtains progesterone by single culture of upstream module strains. The upstream module strains were inoculated in 5 mL seed culture medium, cultured at 30 ° C and 220 rpm for 14 to 16 h, and inoculated in 50 mL YPD fermentation medium with an initial bacterial concentration of OD ₆₀₀ = 0.1, cultured at 28 ° C and 220 rpm, and the bacterial density (OD ₆₀₀ ) and progesterone production during the fermentation process were monitored. The upstream module strains with Vaccinia virus source, type II Homo sapiens source and Bos taurus source were cultured in YPD fermentation medium containing 50 g / L glucose at 28 ° C and 220 rpm for 8 days, and progesterone production of 9.56 mg / L, 9.12 mg / L and 5.53 mg / L were obtained respectively (Figure 12). In this example, the progesterone de novo synthesis ability of 3β-HSD of each source species is not completely consistent with the results of the bioconversion experiment in Example 1, indicating that the catalytic efficiency of 3β-HSD is affected by different reaction conditions and host genotypes. Among them, type II human 3β-HSD (Hs_3β-HSD) showed catalytic advantages in de novo synthesis and whole-cell catalytic synthesis of progesterone experiments.

Example 6: De novo synthesis of 17-hydroxyprogesterone and 17-hydroxypregnenolone using glucose

1. Acquisition of strains

Module A, module B, SyBE_Yl2091025, SyBE_Yl2091006, and SyBE_Yl2091016 are as described in Example 5.

2. Experimental methods

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder;

YPD fermentation medium: 50g/L glucose, 20g/L peptone, 10g/L yeast extract powder.

3. Experimental results

The present invention firstly integrates module A and module B containing genes from the same species into the wild-type Yarrowia lipolytica strain ATCC201249, and obtains strains SyBE_Yl2091006 (containing Mesocricetus auratus genes) and SyBE_Yl2091016 (containing Ovis aries genes) as downstream module strains. Module C described in Example 5 containing genes from different species is respectively integrated into the high-yielding campesterol Yarrowia lipolytica chassis strain SyBE_Yl2060077, and obtains strains SyBE_Yl2091025-SyBE_Yl2091028, SyBE_Yl2090006 and SyBE_Yl2090018 as upstream module strains.

The present invention obtains the target steroid by combined module mixed fermentation. The upstream module strain SyBE_Yl2091025 with Bos taurus source and two downstream module strains SyBE_Yl2091006 and SyBE_Yl2091016 are inoculated in 5 mL seed culture medium respectively, and cultured at 30°C and 220 rpm for 14 to 16 hours. The strain SyBE_Yl2091025 is inoculated with SyBE_Yl2091006 and SyBE_Yl2091016 at an _OD600 ratio of 10:1, and the final _OD600 is 0.1. The culture is carried out at 28°C and 220 rpm for 8 days, and the cell density ( _OD600 ) and steroid yield during the fermentation process are monitored.

In the SyBE_Yl2091025-SyBE_Yl2091006 mixed bacteria system, 0.25 mg/L of 17-hydroxyprogesterone, 0.74 mg/L of 17-hydroxypregnenolone, and the production of androstenedione were 0.88 mg/L. In the SyBE_Yl2091025-SyBE_Yl2091016 mixed bacteria system, 0.91 mg/L of 17-hydroxyprogesterone, 0.29 mg/L of 17-hydroxyprogesterone, and the production of androstenedione were 1.03 mg/L (Figure 13). So far, the present invention has achieved the de novo synthesis of 17-hydroxypregnenolone and 17-hydroxyprogesterone using a mixed bacteria system. The Δ ⁴ steroids (P4, 17OHP4, 4AD) synthesized by the mixed bacteria system account for 56.5 to 83.1% of the total steroid products. The difficult-to-convert intermediate 17OHP5 accounted for only 12.2% to 37.3% of the total steroids, which was much higher than the 17OHP5 proportion in the single-bacteria system (90.4% to 99.1%). These results indicate that the co-culture system design successfully alleviated the substrate competition between 3β-HSD and CYP17A1 by forcing the substrate P5 to be preferentially utilized by 3β-HSD, allowing more steroid flux to be used for Δ ⁴ -steroid synthesis.

Example 7: De novo synthesis of dehydroepiandrosterone using glucose

1. Acquisition of strains

SyBE_Yl2091026 and SyBE_Yl2091006 are as described in Example 5.

2. Experimental methods

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder;

YPD fermentation medium: 50g/L glucose, 20g/L peptone, 10g/L yeast extract powder.

3. Experimental results

The upstream module strain SyBE_Yl2091026 and the downstream module strain SyBE_Yl2091006 were inoculated into 5 mL seed culture medium and cultured at 30°C and 220 rpm for 14-16 h. The strains SyBE_Yl2091026 and SyBE_Yl2091006 were inoculated into 50 mL YPD fermentation medium at an OD ₆₀₀ ratio of 10:1 and a final OD ₆₀₀ = 0.1, and cultured at 28°C and 220 rpm for 8 days, and the cell density (OD ₆₀₀ ) and steroid production during the fermentation process were monitored.

In the SyBE_Y12091026-SyBE_Y12091006 mixed bacteria system, 2.13 mg/L of dehydroepiandrosterone was synthesized (Figure 14). Compared with the mixed bacteria system in Example 6, which used bovine 3β-HSD expression engineering bacteria as the upstream module and tended to synthesize 4AD, the steroid synthesis in the current mixed bacteria system tended to accumulate DHEA, proving that different 3β-HSD source strains have a greater impact on the system steroid synthesis and metabolic flow.

Example 8: De novo synthesis of androstenedione and testosterone using glucose

1. Acquisition of experimental materials

The acquisition of wild-type Yarrowia lipolytica, the construction of modular integration plasmids (modules A to C) and the acquisition of exogenous functional gene elements are the same as described in Example 5.

Construction of module D: The left arm of the IntF integration site, pUC18H after HincII digestion, the leucine nutritional screening tag Leu2 with LoxP sites at both ends, the right arm of the IntD integration site, and the CYP17A1 and POR modules with the same species origin (Ovis aries and Mesocricetus auratus) in Example 5 were spliced together by the Gibson method to obtain a fragment containing NotI digestion sites at both ends, and module D was obtained after NotI digestion.

The acquisition of SyBE_Y12091030 was the same as described in Example 3

The 17-hydroxylation transformation experiment verified the construction of the strain, and the module A containing four genes from different sources was integrated into ATCC201249 to obtain strains SyBE_Yl2091001 to SyBE_Yl2091004.

The construction of the strain was verified by 17,20-lysis transformation experiments. The module B containing three genes from different sources was respectively integrated into the strains SyBE_Yl2091001 to SyBE_Yl2091004 constructed above to obtain strains SyBE_Yl2091005 to SyBE_Yl2091016.

2. Experimental methods

Biotransformation medium: 20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract powder;

Seed culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder;

YPD fermentation medium: 50g/L glucose, 20g/L peptone, 10g/L yeast extract powder.

Steroid substrate stock solution: 1.75 g/L (50% EtOH-Tween80) progesterone, 1.75 g/L (50% EtOH-Tween80) 17-hydroxyprogesterone solution

17-Hydroxylation transformation experiment: SyBE_Yl2091001-SyBE_Yl2091004 were inoculated into 5 mL seed culture medium, cultured at 30°C and 220 rpm for 14-16 h, inoculated into 5 mL biotransformation culture medium with an initial bacterial concentration of _OD600 = 0.1, cultured at 28°C and 220 rpm for 24 h, added 150 μL progesterone substrate mother solution and continued to incubate for 16 h, and took 1 mL sample to detect the 17OHP4 content according to the method in Example 1

17,20-cleavage transformation experiment: SyBE_Yl2091005-SyBE_Yl2091016 were inoculated into 5 mL of seed culture medium and cultured at 30°C and 220 rpm for 14-16 h. They were inoculated into 5 mL of biotransformation medium with an initial cell concentration of _OD600 = 0.1 and cultured at 28°C and 220 rpm for 24 h. 150 μL of 17-hydroxyprogesterone substrate mother solution was added and incubated for another 120 h. 1 mL of sample was taken and the androstenedione content was detected according to the method in Example 1.

Mixed bacteria synthesis of androstenedione experiment: The upstream module strain SyBE_Yl2091025 and the two downstream module strains SyBE_Yl2091016 and SyBE_Yl2091030 were inoculated into 5 mL seed culture medium and cultured at 30°C and 220 rpm for 14-16 h. The upstream module strain and the two downstream module strains were inoculated into 50 mL YPD fermentation medium at an OD ₆₀₀ ratio of 10:1 and a final OD ₆₀₀ of 0.1, and cultured at 28°C and 220 rpm for 8 days to determine the steroid yield.

3. Experimental results

In the biotransformation experiment, the strain containing Ovis aries-derived CYP17A1 showed the strongest 17-hydroxylation ability for progesterone.

As shown in Figure 15 (left), Ec_CYP17A1 and Xl_CYP17A1 have weak catalytic activity for the 17α-hydroxylation of P4. Ma_CYP17A1 and Oa_CYP17A1 have strong activity for the 17α-hydroxylation of P4. Among the tested △ ^{4, 5} type CYP17A1, Ma_CYP17A1 has the strongest activity for the 17α-hydroxylation of P4, but it is only 13.8% of the corresponding activity of △ ⁵ type sheep-derived CYP17A1.

As shown in Figure 15 (right), Ma_CYP17A1 and Ec_CYP17A1 have the strongest 17,20-cleavage activity when using 17OHP4 as substrate. The 17,20-cleavage activity of Oa_CYP17A1 on 17OHP4 depends on the selection of CYB5 source. When Oa_CYB5 and Ec_CYB5 match the above three sources of CYP17A1, CYP17A1 can show strong 17,20-cleavage activity.

As shown in Figure 16, in the 17,20-cleavage conversion experiment, the CYP17A1 and CYB5 from Equus caballus in the current CYP17A1-CYB5 combination showed universal 17,20-cleavage ability. Therefore, in the reconstruction of the downstream strain, the Cre-loxp method was used to remove the Leu2 tag of SyBE_Yl2091004, and the module two and module four with genes from Equus caballus were introduced to obtain the strain SyBE_Yl2091030.

In the SyBE_Yl2091025-SyBE_Yl2091030 mixed fermentation experiment, androstenedione was the main product, and 5.02 mg/L of androstenedione and 1.09 mg/L of testosterone were obtained. In the SyBE_Yl2091025-SyBE_Yl2091030 mixed fermentation experiment, androstenedione increased by 3.9 times compared with the co-culture system before optimization. The above results show that by introducing Equuscaballus-derived Ec_CYP17A1 with strong 17,20-cleavage ability, sheep-derived Oa_CYP17A1 with strong 17α-hydroxylation ability, and horse-derived Ec_CYB5 that can simultaneously promote the 17,20-cleavage ability of Ec_CYP17A1 and Oa_CYP17A1, the substrate conversion efficiency of the downstream pathway can be effectively improved, effectively driving the conversion of steroid intermediates to 4AD.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

1. Expresses the application of any one of the following items in synthesizing steroid compounds and/or steroid hormone medicaments:

(I) CYP17A1 and POR derived from Equus caballus, ovis aries, mesocriticus auratus or Xenopus laevis; and/or

(II) CYB5 derived from Equus caballus, ovis aries, mesocricetus auratus, or Sus scrofa; and/or

(III) 3 β -HSDs derived from Mus musculus, bos taurus, vaccinia virus, arabidopsis thaliana, mycobacterium tuberculosis, homo sapiens (type I), homo sapiens (type II), homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation); and/or (IV), mYP 11A1 from Sus scrofa.

2. A module, comprising any of the following expressions:

(I) CYP17A1 and POR derived from Equus caballus, ovis aries, mesocriticus auratus or Xenopus laevis; and/or

(II) CYB5 derived from Equus caballus, ovis aries or mesocracites auratus or Sus scrofa; and/or

(III) 3 β -HSD derived from Mus musculus, bos taurus, vaccinia virus, arabidopsis thaliana, mycobacterium tuberculosis, homo sapiens (type I), homo sapiens (type II), homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation); and/or

(IV) mYP 11A1 derived from Sus scrofa.

3. A plasmid comprising the expression element of claim 2.

4. A host comprising the plasmid of claim 3.

5. The host of claim 4, comprising one or more of module one, module two, module three, module four, module five, module six, module seven, module A, module B, module C, or module D:

the module one comprises CYP17A1 and POR from Equus caballus, ovis aries, mesocriticus auratus or Xenopus laevis; the first module comprises an IntD integration site and/or a Leu2 label with LoxP sites at two ends; and/or

The second module comprises CYB5 from Equus caballus, ovis aries, mesocriticus auratus or Susscrofa; the second module comprises an IntB integration site and/or a Ura3 tag; and/or

The third module comprises 3 beta-HSD derived from Mus musculus, bos taurus, vaccinia virus, arabidopsis thaliana, mycobacterium tuberculosis, homo sapiens (type I), homo sapiens (type II), homo sapiens (type I, L237S mutation) or Homo sapiens (type II, L236S mutation); the vector connected with the module III comprises pINA1269-Nat; and/or

The module four comprises CYP17A1 and POR from Equus caballus; the fourth module comprises an IntF integration site and/or a Leu2 label with LoxP sites at two ends; and/or

The module five comprises: CYP17A1 and POR from Ovis aries; the module five knocks out the Leu2 tag; and/or

The module six comprises; 3 β -HSD derived from Homo sapiens (type II); the sixth module comprises an IntC integration site and/or a Leu2 label with LoxP sites at two ends; and/or

The module seven comprises: CYP17A1 and POR derived from Mesocriticus auratus, 3 β -HSD derived from Homo sapiens (type II), and 3 β -HSD derived from Vaccidia virus; CYP17A1 and POR comprise an IntD integration site and/or a Leu2 label with LoxP sites at two ends; the 3 beta-HSD linked vector derived from Vaccinium virus comprises pINA1269-Nat; the 3 beta-HSD linked vector derived from Homo sapiens (type II) comprises pUC57-Kan-Simple; and/or

The module A comprises: CYP17A1 and POR derived from Equus caballus, ovis aries, mesocriticus auratus or Xenopus laevis; the module A comprises an IntD integration site and/or a Leu2 label with LoxP sites at two ends; and/or

The module B comprises: CYB5 from Equus caballus, ovis aries or Mesocriticus auratus; said module B comprises an IntB integration site and/or a Ura3 tag; and/or

The module C comprises: 3 β -HSD derived from Mus musculus, bos taurus, vaccinium virus, arabidopsis thaliana, mycobacterium tuberculosis or Homo sapiens (type II) and mYP 11A1 derived from Sus scrofa; the module C-linked vector comprises pINA1269; and/or

The module D comprises: CYP17A1 and POR from Ovis aries and Mesocricetus auratus; the module D comprises an IntF integration site and/or a Leu2 tag with LoxP sites at two ends.

6. The use of any of the following in the synthesis of steroid compounds and/or steroid hormone drugs:

(I) The expression element of claim 2; and/or

(II) the plasmid of claim 3; and/or

(III) the host of claim 4 or 5.

7. The medicine is characterized by comprising any of the following auxiliary materials or auxiliary agents which are acceptable in pharmacy:

(I) The expression element of claim 2; and/or

(II) the plasmid of claim 3; and/or

(III) the host of claim 4 or 5.

8. A pharmaceutical combination comprising the medicament of claim 7 and any other active ingredient.

9. A method for the synthesis of steroid and/or steroid hormone drugs, comprising culturing the host of claim 4 or 5 and collecting the culture.

10. The method of claim 9, comprising:

(I) De novo synthesis of progesterone in a microbial chassis using a carbon source and/or 3 β -HSD; and/or

(II) de novo synthesis of 17-hydroxyprogesterone in a microbial chassis using a carbon source, 3 β -HSD, CYP17A1 and POR; and/or

(III) de novo synthesis of 17-hydroxypregnanolone in a microbial tray using carbon source, CYP17A1 and POR; and/or

(iv) de novo synthesis of dehydroepiandrosterone in a microbial chassis using a carbon source, CYP17A1, POR, and/or CYB5; and/or

(V) synthesizing androstenedione and testosterone de novo on a microbial chassis by using a carbon source, CYP17A1, POR, CYB5 and/or 3 beta-HSD; and/or

(VI) oriented synthesis of androstenedione: co-culturing an upstream module expressing 3 β -HSD in admixture with a downstream module expressing CYP17A1, POR and/or CYB5; and/or

(VII), synthesis of 17-hydroxyprogesterone: co-culturing an upstream module co-expressing the pregnenolone pathway and/or 3 β -HSD in admixture with a downstream module expressing CYP17A1, CYB5 and POR only; and/or

(viii) synthesizing one or more of P4, 17OHP5, 17OHP4 or DHEA using P5 as a substrate; and/or

(ix), synthesizing 17OHP4 using 17OHP5 as a substrate; and/or

(X) synthesizing DHEA using 17OHP5 as a substrate; and/or

(XI) and synthesizing 4AD and TS by using DHEA as a substrate; and/or

(XII) 4AD and TS were synthesized as 17OHP4.

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